Pulsed laser and bioanalytic system

ABSTRACT

Apparatus and methods for producing ultrashort optical pulses are described. A high-power, solid-state, passively mode-locked laser can be manufactured in a compact module that can be incorporated into a portable instrument for biological or chemical analyses. The pulsed laser may produce sub-100-ps optical pulses at a repetition rate commensurate with electronic data-acquisition rates. The optical pulses may excite samples in reaction chambers of the instrument, and be used to generate a reference clock for operating signal-acquisition and signal-processing electronics of the instrument.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application No.62/164,485, filed May 20, 2015, titled “Pulsed Laser,” to U.S.provisional application No. 62/310,398, filed Mar. 18, 2016, titled“Pulsed Laser and System,” to U.S. provisional application No.62/164,482, filed May 20, 2015, titled “Methods for Nucleic AcidSequencing,” and to U.S. provisional application No. 62/289,019, filedJan. 29, 2016, titled “Friction-Drive Electromechanical Motor.” Thisapplication is a continuation-in-part of U.S. application Ser. No.14/821,656, filed Aug. 7, 2015 titled “Integrated Device for TemporalBinning of Received Photons,” and of U.S. application Ser. No.14/821,688, filed Aug. 7, 2015, titled “Integrated Device for Probing,Detecting and Analyzing Molecules.” Each of the foregoing applicationsis hereby incorporated by reference in its entirety.

FIELD

The present application is directed to apparatus and methods forproducing optical pulses and instrumentation for using the opticalpulses to analyze chemical and biological specimens.

BACKGROUND

Ultrashort optical pulses (i.e., optical pulses less than about 100picoseconds) are useful in various areas of research and development aswell as commercial applications involving time-domain analyses. Forexample, ultrashort optical pulses may be useful for time-domainspectroscopy, optical ranging, time-domain imaging (TDI), opticalcoherence tomography (OCT), fluorescent lifetime imaging (FLI), andlifetime-resolved fluorescent detection for genetic sequencing.Ultrashort pulses may also be useful for commercial applicationsincluding optical communication systems, medical applications, andtesting of optoelectronic devices.

Conventional mode-locked lasers have been developed to produceultrashort optical pulses, and a variety of such lasers are currentlyavailable commercially. For example, some solid-state lasers and fiberlasers have been developed to deliver pulses with durations well below200 femtoseconds. However, for some applications, these pulse durationsmay be shorter than is needed and the cost of these lasing systems maybe prohibitively high for certain applications. Additionally, theselasing systems may be stand-alone systems that have a sizeable footprint(e.g., on the order of 1 ft² or larger), and are not readily portable orincorporated into other portable systems as a module.

SUMMARY

The technology described herein relates to apparatus and methods forproducing ultrashort optical pulses. A mode-locked laser system isdescribed that may be implemented as a compact, low-cost laser capableof producing sub-100-picosecond pulses at −100 MHz pulse-repetitionrates. The optical pulses may be delivered to reaction chambers of achemical or bioanalytical system. The optical pulses from the laser maybe detected electronically and the signal processed to produce anelectronic clock signal that synchronizes and drives data-acquisitionelectronics of the system. The inventors have recognized and appreciatedthat a compact, low-cost, pulsed-laser system may be incorporated intoinstrumentation (e.g., time-of-flight imaging instruments, bioanalyticalinstruments that utilize lifetime-resolved fluorescent detection,genetic sequencing instruments, optical coherence tomographyinstruments, etc.), and may allow such instrumentation to become readilyportable and produced at appreciably lower cost than is the case forconventional instrumentation requiring an ultrashort pulsed laser. Highportability may make such instruments more useful for research,development, clinical use, field deployment, and commercialapplications.

Some embodiments relate to a mode-locked laser comprising a base platehaving a maximum edge length of not more than 350 mm, a gain mediummounted on the base plate, a first end mirror mounted on the base platelocated at a first end of a laser cavity, and a saturable-absorbermirror mounted on the base plate and forming a second end mirror for thelaser cavity, wherein the mode-locked laser is configured to produceoptical pulses by passive mode locking at a repetition rate between 50MHz and 200 MHz

Some embodiments relate to a method for sequencing DNA. The method maycomprise acts of producing pulsed excitation energy at a singlecharacteristic wavelength, directing the pulsed excitation energytowards a bio-optoelectronic chip, wherein the bio-optoelectronic chipsupports sequential incorporation of nucleotides or nucleotide analogsinto a growing strand that is complementary to a target nucleic acid,receiving signals representative of fluorescent emission induced by thepulsed excitation energy at the single characteristic wavelength,wherein the signals correspond to the sequential incorporation ofnucleotides or nucleotide analogs into the growing strand, andprocessing the received signals to determine the identity of fourdifferent nucleotides or nucleotide analogs incorporated into thegrowing strand.

Some embodiments relate to a bioanalytic instrument comprising a pulsedlaser system configured to produce optical excitation pulses at a singlecharacteristic wavelength, a receptacle for receiving abio-optoelectronic chip and making electrical connections and an opticalcoupling with the bio-optoelectronic chip, wherein thebio-optoelectronic chip supports sequential incorporation of nucleotidesor nucleotide analogs into a growing strand that is complementary to atarget nucleic acid, beam-steering optics arranged to direct theexcitation pulses towards the receptacle, and a signal processorconfigured to receive signals representative of fluorescent emissioninduced by the excitation pulses at the single characteristic wavelengthand process the received signals to determine the identity of fourdifferent nucleotides or nucleotide analogs incorporated into thegrowing strand, wherein the received signals correspond to thesequential incorporation of nucleotides or nucleotide analogs into thegrowing strand.

Some embodiments relate to bioanalytic instrument comprising a laserconfigured to produce pulsed excitation energy at a singlecharacteristic wavelength and a clock-generation circuit configured tosynchronize a first clock signal from an electronic orelectro-mechanical oscillator to a second clock signal produced fromdetection of optical pulses from the laser and to provide thesynchronized first clock signal to time data-acquisition by thebioanalytic instrument.

Some embodiments relate to a system comprising a pulsed laser, acontinuous-wave laser, a first nonlinear optical element, and a secondnonlinear optical element, wherein the system is configured to produce afirst pulse train generated from the first nonlinear optical element ata first characteristic wavelength and a second pulse train from thesecond nonlinear optical element at a second characteristic wavelength.

Some embodiments relate to a method of providing synchronized opticalpulses. The method may include acts of operating a pulsed laser at afirst characteristic wavelength, operating a continuous-wave laser at asecond characteristic wavelength, coupling a first pulse train from thepulsed laser into a laser cavity of the continuous-wave laser, andgenerating a second pulse train at a third characteristic wavelength inthe laser cavity of the continuous-wave laser.

Some embodiments relate to a system comprising a first pulsed laser, asecond pulsed laser, a first nonlinear optical element, and a secondnonlinear optical element, wherein the system is configured to produce afirst pulse train generated from the first nonlinear optical element ata first characteristic wavelength and a second pulse train bysum-frequency generation from the second nonlinear optical element at asecond characteristic wavelength.

Some embodiments relate to a method of providing synchronized opticalpulses. The method may include acts of operating a first pulsed laser ata first characteristic wavelength, operating a second pulsed laser at asecond characteristic wavelength, synchronizing the first pulsed laserto the second pulsed laser, frequency doubling pulses from the firstpulsed laser to produce a first pulse train at a third characteristicwavelength, coupling pulses from the first pulsed laser and secondpulsed laser into a nonlinear optical element, and generating, bysum-frequency generation, a second pulse train at a fourthcharacteristic wavelength.

Some embodiments relate to a system comprising a first pulsed laser anda second pulsed laser that includes an intracavity saturable absorbermirror, wherein the system is configured to direct pulses from the firstpulsed laser onto the saturable absorber mirror of the second pulsedlaser.

Some embodiments relate to a method for mode locking two lasers. Themethod may include acts of operating a first pulsed laser at a firstcharacteristic wavelength and coupling a pulse train from the firstpulsed laser onto a saturable absorber mirror in a laser cavity of asecond pulsed laser.

Some embodiments relate to a pulsed laser system comprising a firstmode-locked laser having a first laser cavity configured to producepulses having a first characteristic wavelength at a first repetitionrate, a second laser having a second laser cavity configured to producecontinuous-wave radiation, a nonlinear optical element within the secondlaser cavity, and optical elements that direct an output from the firstmode-locked laser into the nonlinear optical element.

Some embodiments relate to a method of producing optical pulses atmultiple characteristic wavelengths. The method may include acts ofproducing optical pulses in a first mode-locked laser having a firstlaser cavity at a first characteristic wavelengths, operating a secondlaser having a second laser cavity in continuous-wave mode at a secondcharacteristic wavelengths, injecting pulses from the first mode-lockedlaser into a nonlinear optical element in the second laser cavity, andgenerating, by sum-frequency generation, optical pulses in the nonlinearoptical element at a third characteristic wavelengths

Some embodiments relate to a pulsed laser comprising a base structure, adiode pump source mounted within the base structure, and a laser cavitywithin the base structure that includes a gain medium and is configuredto produce optical pulses, wherein the diode pump source and gain mediumare each mounted on a platform that is partially thermally andmechanically isolated from the base structure.

The foregoing and other aspects, implementations, acts, functionalities,features and, embodiments of the present teachings can be more fullyunderstood from the following description in conjunction with theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the figures, described herein,are for illustration purposes only. It is to be understood that in someinstances various aspects of the invention may be shown exaggerated orenlarged to facilitate an understanding of the invention. In thedrawings, like reference characters generally refer to like features,functionally similar and/or structurally similar elements throughout thevarious figures. The drawings are not necessarily to scale, emphasisinstead being placed upon illustrating the principles of the teachings.The drawings are not intended to limit the scope of the presentteachings in any way.

FIG. 1-1A is a block diagram depiction of an analytical instrument,according to some embodiments.

FIG. 1-1B depicts a pulsed laser incorporated into an analyticalinstrument, according to some embodiments.

FIG. 1-2 depicts a train of optical pulses, according to someembodiments.

FIG. 1-3 depicts an example of parallel reaction chambers that may beexcited optically by a pulsed laser via one or more waveguides andcorresponding detectors for each chamber, according to some embodiments.

FIG. 1-4 illustrates optical excitation of a reaction chamber from awaveguide, according to some embodiments.

FIG. 1-5 depicts further details of an integrated reaction chamber,optical waveguide, and time-binning photodetector, according to someembodiments.

FIG. 1-6 depicts an example of a biological reaction that may occurwithin a reaction chamber, according to some embodiments.

FIG. 1-7 depicts emission probability curves for two differentfluorophores having different decay characteristics.

FIG. 1-8 depicts time-binning detection of fluorescent emission,according to some embodiments.

FIG. 1-9 depicts a time-binning photodetector, according to someembodiments.

FIG. 1-10A depicts pulsed excitation and time-binned detection offluorescent emission from a sample, according to some embodiments.

FIG. 1-10B depicts a histogram of accumulated fluorescent photon countsin various time bins after repeated pulsed excitation of a sample,according to some embodiments.

FIG. 1-11A-1-11D depict different histograms that may correspond to thefour nucleotides (T, A, C, G) or nucleotide analogs, according to someembodiments.

FIG. 2-1A depicts a pulsed laser system, according to some embodiments.

FIG. 2-1B depicts a pulsed laser system incorporated into a portableinstrument, according to some embodiments.

FIG. 2-2A depicts an integrated optical mount, according to someembodiments.

FIG. 2-2B depicts an optic mounted in an integrated optical mount,according to some embodiments.

FIG. 3-1 depicts a diode-pumped, solid-state, mode-locked laser,according to some embodiments.

FIG. 3-2A through FIG. 3-2D depict various embodiments ofoptical-path-length extenders which may be incorporated as part of alaser cavity, according to some implementations.

FIG. 3-3A depicts a diode-pumped, solid-state, mode-locked laser, forwhich frequency doubling is external to the laser cavity, according tosome embodiments.

FIG. 3-3B depicts a diode-pumped, solid-state, nonlinear-mirrormode-locked laser, according to some embodiments.

FIG. 3-3C depicts a diode-pumped, solid-state, multi-wavelength,mode-locked laser, according to some embodiments.

FIG. 3-4A depicts a portion of a saturable-absorber mirror, according tosome implementations.

FIG. 3-4B depicts a band-gap diagram for the saturable-absorber mirrorof FIG. 3-4A, according to some embodiments.

FIG. 3-4C illustrates intensity profiles at the locations of quantumwell absorbers in a saturable-absorber mirror, according to someembodiments.

FIG. 3-5A depicts an output coupler for a multi-wavelength, mode-lockedlaser, according to some embodiments.

FIG. 3-5B depicts an output coupler for a multi-wavelength, mode-lockedlaser, according to some embodiments.

FIG. 3-6 illustrates a mount for a gain medium or other high-poweroptical component which may be used in a compact mode-locked laser,according to some embodiments.

FIG. 3-7A depicts, in plan view, a platform for mounting a gain mediumor other high-power optical system which may be used in a compactmode-locked laser, according to some embodiments.

FIG. 3-7B and FIG. 3-7C depict elevation views of the platformillustrated in FIG. 3-7A, according to some embodiments.

FIG. 3-8A depicts a two-laser system for producing synchronized pulsetrains at two wavelengths in which one laser operates in acontinuous-wave mode, according to some embodiments.

FIG. 3-8B depicts a two-laser system for producing synchronized pulsetrains at two wavelengths in which one laser operates in acontinuous-wave mode, according to some embodiments.

FIG. 3-9 depicts a two-laser system for producing synchronized pulsetrains at two wavelengths in which one laser partially bleaches asaturable absorber of a second laser, according to some embodiments.

FIG. 3-10 depicts an electro-mechanical control circuit for controllinga laser cavity length in a synchronized laser system, according to someembodiments.

FIG. 4-1 and FIG. 4-2 depict mode-locked, laser diodes, according tosome embodiments.

FIG. 4-3 depicts a mode-locked, laser diode that includes a length ofoptical fiber as an optical delay element, according to someimplementations.

FIG. 5-1 through FIG. 5-3 depict mode-locked fiber lasers, according tosome embodiments.

FIG. 6-1A illustrates optical pump and output pulses for gain switching,according to some embodiments.

FIG. 6-1B illustrates relaxation oscillations, according to someembodiments.

FIG. 6-1C depicts an optical output pulse showing a tail, according tosome embodiments.

FIG. 6-2A depicts a pulsed semiconductor laser diode, according to someembodiments.

FIG. 6-2B depicts a pulser circuit schematic for pulsing a laser diodeor light-emitting diode, according to one embodiment.

FIG. 6-2C illustrates improvements in current delivered to a laserdiode, according to some embodiments.

FIG. 6-3 depicts a drive waveform for gain-switching a laser diode,according to some embodiments.

FIG. 6-4A depicts a pulser circuit for driving a laser diode orlight-emitting diode, in some embodiments.

FIG. 6-4B depicts a pulser circuit schematic for driving a laser diodeor light-emitting diode, according to some embodiments.

FIG. 6-4C depicts a pulser circuit schematic for driving a laser diodeor light-emitting diode, according to some embodiments.

FIG. 6-4D depicts an RF driver for pulsing a laser diode orlight-emitting diode, according to some embodiments.

FIG. 6-4E illustrates a drive waveform produced by the circuit of FIG.6-4D, according to some embodiments.

FIG. 6-4F depicts an RF driver for pulsing a laser diode orlight-emitting diode, according to some embodiments.

FIG. 6-4G illustrates drive waveforms produced by the circuit of FIG.6-4F, according to some embodiments.

FIG. 6-4H depicts a pulser circuit schematic for driving a laser diodeor light-emitting diode, according to some embodiments.

FIG. 6-41 illustrates efficiency of power coupling to a laser diode,according to some embodiments.

FIG. 6-4J depicts a pulser and driver circuit for pulsing opticalemission from a laser diode or light-emitting diode, according to someembodiments.

FIG. 6-4K depicts a pulser circuit for producing a train of pulses,according to some embodiments.

FIG. 6-4L illustrates data inputs to a logic gate in a pulser circuit,according to some embodiments.

FIG. 6-4M depicts a driver circuit for driving a laser diode orlight-emitting diode with electrical pulses, according to someembodiments.

FIG. 6-5A depicts a pulser circuit for gain-switching a laser diode,according to some embodiments.

FIG. 6-5B illustrates a drive voltage from a pulser circuit, accordingto some embodiments.

FIG. 6-5C and FIG. 6-5D illustrate example measurements of ultrafastoptical pulses produced from a gain-switched laser diode, according tosome embodiments.

FIG. 6-6A depicts a slab-coupled optical waveguide semiconductor laserthat may be gain-switched or Q-switched, according to some embodiments.

FIG. 6-6B illustrates an optical mode profile in a slab-coupled opticalwaveguide laser, according to some embodiments.

FIG. 6-6C depicts an integrated, gain-switched semiconductor laser andcoupled saturable absorber, according to some embodiments.

FIG. 7-1A depicts an optical switch array configured to produce pulsesfrom a continuous-wave laser, according to some embodiments.

FIG. 7-1B illustrates driving waveforms for switches of the opticalswitch array depicted in FIG. 7-1A, according to some implementations.

FIG. 7-1C depicts optical intensities in several ports of the opticalswitch array depicted in FIG. 7-1A, according to some implementations.

FIG. 7-1D illustrates driving waveforms for switches of the opticalswitch array depicted in FIG. 7-1A, according to some implementations.

FIG. 7-1E depicts optical intensities in several ports of the opticalswitch array depicted in FIG. 7-1A, according to some implementations.

FIG. 8-1 depicts a beam-steering module, according to some embodiments.

FIG. 8-2 depicts optical details of a beam-steering module, according tosome embodiments.

FIG. 8-3 depicts alignment of a pulsed-laser beam to an optical coupleron a chip, according to some embodiments.

FIG. 8-4 depicts detection and control circuitry for coupling opticalpulses from a pulsed laser into multiple waveguides of abio-optoelectronic chip, according to some embodiments.

FIG. 8-5 depicts acts associated with methods of coupling optical pulsesfrom a pulsed laser into multiple waveguides of a bio-optoelectronicchip, according to some embodiments.

FIG. 9-1 depicts a system for synchronizing timing of optical pulses toinstrument electronics, according to some embodiments.

FIG. 9-2 depicts a system for synchronizing timing of optical pulses toinstrument electronics, according to some embodiments.

FIG. 9-3 depicts clock-generation circuitry for an analytical instrumentthat incorporates a pulsed optical source, according to someembodiments.

FIG. 9-4 depicts a system for synchronizing timing of optical pulsesfrom two pulse sources to instrument electronics, according to someembodiments.

FIG. 9-5A depicts a system for synchronizing interleaved timing ofoptical pulses from two pulse sources to instrument electronics,according to some embodiments.

FIG. 9-5B depicts interleaved and synchronized pulse trains from twopulsed optical sources, according to some embodiments.

FIG. 9-6A depicts a two-laser system for producing synchronized pulsetrains at two or more wavelengths, according to some embodiments.

FIG. 9-6B depicts a two-laser system for producing synchronized pulsetrains at two wavelengths, according to some embodiments.

The features and advantages of the present invention will become moreapparent from the detailed description set forth below when taken inconjunction with the drawings. When describing embodiments in referenceto the drawings, directional references (“above,” “below,” “top,”“bottom,” “left,” “right,” “horizontal,” “vertical,” etc.) may be used.Such references are intended merely as an aid to the reader viewing thedrawings in a normal orientation. These directional references are notintended to describe a preferred or only orientation of features of anembodied device. A device may be embodied using other orientations.

DETAILED DESCRIPTION I. Introduction

The inventors have recognized and appreciated that conventionalultrashort pulsed lasers are typically large, expensive, and unsuitablefor many mobile applications and/or incorporating into portableinstrumentation that may be adapted for imaging, ranging, orbioanalytical applications. Accordingly, the inventors have conceived ofcompact, ultrashort-pulsed lasing systems that can providesub-100-picosecond pulses at selected wavelengths and at average opticalpowers up to ˜400 milliwatts (mW). The lasing system may be configuredto provide a repetition rate of optical pulses between about 50 MHz andabout 200 MHz. In some embodiments, an area occupied by a pulsed laserand its optics may be about the size of an A4 sheet of paper with athickness of about 40 mm or less. In some implementations, a pulsedsemiconductor laser may be substantially smaller than this size.

The term “optical” may refer to ultra-violet, visible, near-infrared,and short-wavelength infrared spectral bands.

In some bioanalytic applications, such as genetic sequencing ormassively-parallel assays, a compact pulsed lasing system may be used todeliver optical excitation energy to a plurality of reaction chambersintegrated onto a chip. The number of reaction chambers on the chip maybe between about 10,000 and about 10,000,000, and the chambers maycontain samples that may undergo multiple biochemical reactions over aperiod of time, according to some implementations. In otherimplementations, there may be fewer or more reaction chambers on thechip. According to some embodiments, the samples or moleculesinteracting with the samples may be labeled with one or morefluorophores that fluoresce(s), or the samples may fluoresce themselves,following excitation by an optical pulse from a pulsed laser. Detectionand analysis of fluorescence from the reaction chambers providesinformation about the samples within the chambers.

To make a portable instrument that includes such a large number ofreaction chambers and that uses multiple different fluorophores, thereexist several technical challenges. A pulsed lasing system must be smalland lightweight, and it must provide enough optical power to excitefluorophores in all the reaction chambers. Additionally, there must besome way to excite different fluorophores with the pulsed laser (e.g.,four fluorophores with different emission characteristics for DNAsequencing), and detect different emission characteristics at eachreaction chamber from the fluorophores so that each fluorophore can bedistinguished from the other fluorophores.

In overview, an analytical instrument 1-100 may comprise one or morepulsed lasers 1-110 mounted within or otherwise coupled to theinstrument, as depicted in FIG. 1-1A. According to some embodiments, apulsed laser 1-110 may be a mode-locked laser. A mode-locked laser mayinclude an element (e.g., saturable absorber, acoustooptic modulator,Kerr lens) in the laser cavity, or coupled to the laser cavity, thatinduces phase locking of the laser's longitudinal frequency modes. Inother embodiments, a pulsed laser 1-110 may be a gain-switched laser. Again-switched laser may comprise an external modulator that modulatesoptical gain in the laser's gain medium.

The instrument 1-100 may include an optical system 1-115 and an analyticsystem 1-160. The optical system 1-115 may include one or more opticalcomponents (e.g., lens, mirror, optical filter, attenuator) and beconfigured to operate on and/or deliver optical pulses 1-122 from thepulsed laser 1-110 to the analytic system 1-160. The analytic system mayinclude many components that are arranged to direct the optical pulsesto at least one sample that is to be analyzed, receive one or moreoptical signals (e.g., fluorescence, backscattered radiation) from theat least one sample, and produce one or more electrical signalsrepresentative of the received optical signals. In some embodiments, theanalytic system 1-160 may include one or more photodetectors andsignal-processing electronics (e.g., one or more microcontrollers, oneor more field-programmable gate arrays, one or more microprocessors, oneor more digital signal processors, logic gates, etc.) configured toprocess the electrical signals from the photodetectors, and may alsoinclude data transmission hardware configured to transmit and receivedata to and from external devices via a data communications link. Insome embodiments, the analytic system 1-160 may be configured to receivea bio-optoelectronic chip 1-140, which holds one or more samples to beanalyzed.

Although the optical pulses 1-122 are depicted as having a singletransverse optical mode, in some embodiments, the optical output fromthe pulsed laser 1-110 may be multimodal. For example, a transverseoutput beam profile may have multiple intensity peaks and minima due tomultimodal operation of the laser. In some embodiments, a multimodaloutput may be homogenized (e.g., by diffusing optics) when coupled tothe analytic system 1-160. In some implementations, a multimodal outputmay be coupled to a plurality of fibers or waveguides in the analyticsystem 1-160. For example, each intensity peak of a multimodal outputmay be coupled to a separate waveguide that connects to thebio-optoelectronic chip 1-140. Allowing a pulsed laser to operate in amultimode state may enable higher output powers from the pulsed laser.

FIG. 1-1B depicts a further detailed example of an analytical instrument1-100 that includes a pulsed laser 1-110, which may be mounted to aninstrument chassis or frame 1-102 of the instrument. The analyticinstrument may be configured to receive a removable, packaged,bio-optoelectronic chip 1-140. The chip may include a plurality ofreaction chambers, integrated optical components arranged to deliveroptical excitation energy to the reaction chambers, and integratedphotodetectors arranged to detect fluorescent emission from the reactionchambers. In some implementations, the chip 1-140 may be disposable,whereas in other implementations the chip may be reusable. When the chipis received by the instrument, it may be in electrical and opticalcommunication with the pulsed laser and electrical and opticalcommunication with the analytic system 1-160.

In some embodiments, the bio-optoelectronic chip may be mounted (e.g.,via a socket connection) on an electronic circuit board 1-130, such as aprinted circuit board (PCB) that may include additional instrumentelectronics. For example, the PCB 1-130 may include circuitry configuredto provide electrical power, one or more clock signals, and controlsignals to the bio-optoelectronic chip 1-140, and signal-processingcircuitry arranged to receive signals representative of fluorescentemission detected from the reaction chambers. The PCB 1-130 may alsoinclude circuitry configured to receive feedback signals relating tooptical coupling and power levels of the optical pulses 1-122 coupledinto waveguides of the bio-optoelectronic chip 1-140. Data returned fromthe bio-optoelectronic chip may be processed in part or entirely by theinstrument, although data may be transmitted via a network connection toone or more remote data processors, in some implementations.

According to some embodiments, an ultrashort pulsed laser 1-110 maycomprise a gain medium 1-105 (which may be solid-state material in someembodiments), a pump source (e.g., a laser diode, not shown) forexciting the gain medium, an output coupler 1-111, and a laser-cavityend mirror 1-119. The laser's optical cavity may be bound by the outputcoupler and end mirror. An optical axis 1-125 of the laser cavity mayhave one or more folds (turns) to increase the length of the lasercavity. In some embodiments, there may be additional optical elements(not shown) in the laser cavity for beam shaping, wavelength selection,and/or pulse forming. In some cases, the end mirror 1-119 may comprise asaturable-absorber mirror (SAM) that induces passive mode locking oflongitudinal cavity modes and results in pulsed operation of the laser1-110.

When passively mode locked, an intracavity pulse 1-120 may circulatebetween the end mirror 1-119 and the output coupler 1-111, and a portionof the intracavity pulse may be transmitted through the output coupler1-111 as an output pulse 1-122. Accordingly, a train of output pulses1-122, as depicted in the graph of FIG. 1-2, may be detected at theoutput coupler as the intracavity pulse 1-120 bounces back-and-forthbetween the output coupler 1-111 and end mirror 1-119 in the lasercavity.

FIG. 1-2 depicts temporal intensity profiles of the output pulses 1-122.In some embodiments, the peak intensity values of the emitted pulses maybe approximately equal, and the profiles may have a Gaussian temporalprofile, though other profiles such as a sech² profile may be possible.In some cases, the pulses may not have symmetric temporal profiles andmay have other temporal shapes. The duration of each pulse may becharacterized by a full-width-half-maximum (FWHM) value, as indicated inFIG. 1-2. According to some embodiments of a pulsed laser, ultrashortoptical pulses may have FWHM values less than 100 picoseconds (ps). Insome cases, the FWHM values may be less than 30 ps.

The output pulses 1-122 may be separated by regular intervals T. In someembodiment (e.g., for mode-locked lasers), T may be determined by around-trip travel time between the output coupler 1-111 and cavity endmirror 1-119. According to some embodiments, the pulse-separationinterval T may be between about 1 ns and about 30 ns. In some cases, thepulse-separation interval T may be between about 5 ns and about 20 ns,corresponding to a laser-cavity length (an approximate length of theoptical axis 1-125 within the laser cavity) between about 0.7 meter andabout 3 meters.

According to some embodiments, a desired pulse-separation interval T andlaser-cavity length may be determined by a combination of the number ofreaction chambers on the chip 1-140, fluorescent emissioncharacteristics, and the speed of data-handling circuitry for readingdata from the bio-optoelectronic chip 1-140. The inventors haverecognized and appreciated that different fluorophores may bedistinguished by their different fluorescent decay rates. Accordingly,there needs to be sufficient pulse-separation interval T to collectadequate statistics for the selected fluorophores to distinguish betweentheir different decay rates. Additionally, if the pulse-separationinterval T is too short, the data handling circuitry cannot keep up withthe large amount of data being collected by the large number of reactionchambers. The inventors have recognized and appreciated that apulse-separation interval T between about 5 ns and about 20 ns issuitable for fluorophores that have decay rates up to about 2 ns and forhandling data from between about 60,000 and 600,000 reaction chambers.

According to some implementations, a beam-steering module 1-150 mayreceive output pulses from the pulsed laser 1-110 and be configured toadjust the position and incident angles of the optical pulses onto anoptical coupler of the bio-optoelectronic chip 1-140. According to someembodiments, the output pulses from the pulsed laser may be operated onby a beam-steering module 1-150, which is configured to align the beamof output pulses to an optical coupler on the bio-optoelectronic chip1-140. The beam-steering module may provide position and incident angleadjustments for the optical beam at the optical coupler. In someimplementations, the beam-steering module may further provide focusingof the beam of output pulses onto the optical coupler.

Referring to FIG. 1-3, the output pulses 1-122 may be coupled into oneor more optical waveguides 1-312 on the bio-optoelectronic chip. In someembodiments, the optical pulses may be coupled to one or more waveguidesvia a grating coupler 1-310, though coupling to an end of an opticalwaveguide on the bio-optoelectronic chip may be used in someembodiments. According to some embodiments, a quad detector 1-320 may belocated on a semiconductor substrate 1-305 (e.g., a silicon substrate)for aiding in alignment of the beam of optical pulses 1-122 to a gratingcoupler 1-310. The one or more waveguides 1-312 and reaction chambers1-330 may be integrated on the same semiconductor substrate withintervening dielectric layers (e.g., silicon dioxide layers) between thesubstrate, waveguide, reaction chambers, and photodetectors 1-322.

Each waveguide 1-312 may include a tapered portion 1-315 below thereaction chambers 1-330 to equalize optical power coupled to thereaction chambers along the waveguide. The reducing taper may force moreoptical energy outside the waveguide's core, increasing coupling to thereaction chambers and compensating for optical losses along thewaveguide, including losses for light coupling into the reactionchambers. A second grating coupler 1-317 may be located at an end ofeach waveguide to direct optical energy to an integrated photodiode1-324. The integrated photodiode may detect an amount of power coupleddown a waveguide and provide a detected signal to feedback circuitrythat controls the beam-steering module 1-150, for example.

The reaction chambers 1-330 may be aligned with the tapered portion1-315 of the waveguide and recessed in a tub 1-340. There may betime-binning photodetectors 1-322 located on the semiconductor substrate1-305 for each reaction chamber 1-330. A metal coating and/or multilayercoating 1-350 may be formed around the reaction chambers and above thewaveguide to prevent optical excitation of fluorophores that are not inthe reaction chambers (e.g., dispersed in a solution above the reactionchambers). The metal coating and/or multilayer coating 1-350 may beraised beyond edges of the tub 1-340 to reduce absorptive losses of theoptical energy in the waveguide 1-312 at the input and output ends ofeach waveguide.

There may be a plurality of rows of waveguides, reaction chambers, andtime-binning photodetectors on the bio-optoelectronic chip 1-140. Forexample, there may be 128 rows, each having 512 reaction chambers, for atotal of 65,536 reaction chambers in some implementations. Otherimplementations may include fewer or more reaction chambers, and mayinclude other layout configurations. Optical power from the pulsed laser1-110 may be distributed to the multiple waveguides via one or more starcouplers or multi-mode interference couplers, or by any other means,located between an optical coupler to the chip 1-140 and the pluralityof waveguides.

FIG. 1-4 illustrates optical energy coupling from an optical pulse 1-122within a waveguide 1-315 to a reaction chamber 1-330. The drawing hasbeen produced from an electromagnetic field simulation of the opticalwave that accounts for waveguide dimensions, reaction chamberdimensions, the different materials' optical properties, and thedistance of the waveguide 1-315 from the reaction chamber 1-330. Thewaveguide may be formed from silicon nitride in a surrounding medium1-410 of silicon dioxide, for example. The waveguide, surroundingmedium, and reaction chamber may be formed by microfabrication processesdescribed in U.S. application Ser. No. 14/821,688, filed Aug. 7, 2015,titled “Integrated Device for Probing, Detecting and AnalyzingMolecules”. According to some embodiments, an evanescent optical field1-420 couples optical energy transported by the waveguide to thereaction chamber 1-330.

A non-limiting example of a biological reaction taking place in areaction chamber 1-330 is depicted in FIG. 1-5. In this example,sequential incorporation of nucleotides or nucleotide analogs into agrowing strand that is complementary to a target nucleic acid is takingplace in the reaction chamber. The sequential incorporation can bedetected to sequence DNA. The reaction chamber may have a depth betweenabout 150 nm and about 250 nm and a diameter between about 80 nm andabout 160 nm. A metallization layer 1-540 (e.g., a metallization for anelectrical reference potential) may be patterned above the photodetectorto provide an aperture that blocks stray light from adjacent reactionchambers and other unwanted light sources. According to someembodiments, polymerase 1-520 may be located within the reaction chamber1-330 (e.g., attached to a base of the chamber). The polymerase may takeup a target nucleic acid 1-510 (e.g., a portion of nucleic acid derivedfrom DNA), and sequence a growing strand of complementary nucleic acidto produce a growing strand of DNA 1-512. Nucleotides or nucleotideanalogs labeled with different fluorophores may be dispersed in asolution above and within the reaction chamber.

When a labeled nucleotide or nucleotide analog 1-610 is incorporatedinto a growing strand of complementary nucleic acid, as depicted in FIG.1-6, one or more attached fluorophores 1-630 may be repeatedly excitedby pulses of optical energy coupled into the reaction chamber 1-330 fromthe waveguide 1-315. In some embodiments, the fluorophore orfluorophores 1-630 may be attached to one or more nucleotides ornucleotide analogs 1-610 with any suitable linker 1-620. Anincorporation event may last for a period of time up to about 100 ms.During this time, pulses of fluorescent emission resulting fromexcitation of the fluorophore(s) may be detected with a time-binningphotodetector 1-322. By attaching fluorophores with different emissioncharacteristics (e.g., fluorescent decay rates, intensity, fluorescentwavelength) to the different nucleotides (A,C,G,T), detecting anddistinguishing the different emission characteristics while the strandof DNA 1-512 incorporates a nucleic acid and enables determination ofthe genetic sequence of the growing strand of DNA.

According to some embodiments, analytical instrument 1-100 configured toanalyze samples based on fluorescent emission characteristics may detectdifferences in fluorescent lifetimes and/or intensities betweendifferent fluorescent molecules, and/or differences between lifetimesand/or intensities of the same fluorescent molecules in differentenvironments. By way of explanation, FIG. 1-7 plots two differentfluorescent emission probability curves (A and B), which may berepresentative of fluorescent emission from two different fluorescentmolecules, for example. With reference to curve A (dashed line), afterbeing excited by a short or ultrashort optical pulse, a probabilityp_(A)(t) of a fluorescent emission from a first molecule may decay withtime, as depicted. In some cases, the decrease in the probability of aphoton being emitted over time may be represented by an exponentialdecay function p_(A)(t)=P_(Ao)e^(−t/τA), where P_(Ao) is an initialemission probability and TA is a temporal parameter associated with thefirst fluorescent molecule that characterizes the emission decayprobability. τ_(A) may be referred to as the “fluorescence lifetime,”“emission lifetime,” or “lifetime” of the first fluorescent molecule. Insome cases, the value of TA may be altered by a local environment of thefluorescent molecule. Other fluorescent molecules may have differentemission characteristics than that shown in curve A. For example,another fluorescent molecule may have a decay profile that differs froma single exponential decay, and its lifetime may be characterized by ahalf-life value or some other metric.

A second fluorescent molecule may have a decay profile that isexponential, but has a measurably different lifetime TB, as depicted forcurve B in FIG. 1-7. In the example shown, the lifetime for the secondfluorescent molecule of curve B is shorter than the lifetime for curveA, and the probability of emission is higher sooner after excitation ofthe second molecule than for curve A. Different fluorescent moleculesmay have lifetimes or half-life values ranging from about 0.1 ns toabout 20 ns, in some embodiments.

The inventors have recognized and appreciated that differences influorescent emission lifetimes can be used to discern between thepresence or absence of different fluorescent molecules and/or to discernbetween different environments or conditions to which a fluorescentmolecule is subjected. In some cases, discerning fluorescent moleculesbased on lifetime (rather than emission wavelength, for example) cansimplify aspects of an analytical instrument 1-100. As an example,wavelength-discriminating optics (such as wavelength filters, dedicateddetectors for each wavelength, dedicated pulsed optical sources atdifferent wavelengths, and/or diffractive optics) may be reduced innumber or eliminated when discerning fluorescent molecules based onlifetime. In some cases, a single pulsed optical source operating at asingle characteristic wavelength may be used to excite differentfluorescent molecules that emit within a same wavelength region of theoptical spectrum but have measurably different lifetimes. An analyticsystem that uses a single pulsed optical source, rather than multiplesources at different wavelengths, to excite and discern differentfluorescent molecules emitting in a same wavelength region can be lesscomplex to operate and maintain, more compact, and may be manufacturedat lower cost.

Although analytic systems based on fluorescent lifetime analysis mayhave certain benefits, the amount of information obtained by an analyticsystem and/or detection accuracy may be increased by allowing foradditional detection techniques. For example, some analytic systems1-160 may additionally be configured to discern one or more propertiesof a sample based on fluorescent wavelength and/or fluorescentintensity.

Referring again to FIG. 1-7, according to some embodiments, differentfluorescent lifetimes may be distinguished with a photodetector that isconfigured to time-bin fluorescent emission events following excitationof a fluorescent molecule. The time binning may occur during a singlecharge-accumulation cycle for the photodetector. A charge-accumulationcycle is an interval between read-out events during whichphoto-generated carriers are accumulated in bins of the time-binningphotodetector. The concept of determining fluorescent lifetime bytime-binning of emission events is introduced graphically in FIG. 1-8.At time t_(e) just prior to t₁, a fluorescent molecule or ensemble offluorescent molecules of a same type (e.g., the type corresponding tocurve B of FIG. 1-7) is (are) excited by a short or ultrashort opticalpulse. For a large ensemble of molecules, the intensity of emission mayhave a time profile similar to curve B, as depicted in FIG. 1-8.

For a single molecule or a small number of molecules, however, theemission of fluorescent photons occurs according to the statistics ofcurve B in FIG. 1-7, for this example. A time-binning photodetector1-322 may accumulate carriers generated from emission events intodiscrete time bins (three indicated in FIG. 1-8) that are temporallyresolved with respect to the excitation time of the fluorescentmolecule(s). When a large number of emission events are summed, theresulting time bins may approximate the decaying intensity curve shownin FIG. 1-8, and the binned signals can be used to distinguish betweendifferent fluorescent molecules or different environments in which afluorescent molecule is located.

Examples of a time-binning photodetector 1-322 are described in U.S.patent application Ser. No. 14/821,656, filed Aug. 7, 2015, titled“Integrated Device for Temporal Binning of Received Photons,” which isincorporated herein by reference. For explanation purposes, anon-limiting embodiment of a time-binning photodetector is depicted inFIG. 1-9. A single time-binning photodetector 1-900 may comprise aphoton-absorption/carrier-generation region 1-902, a carrier-travelregion 1-906, and a plurality of carrier-storage bins 1-908 a, 1-908 b,1-908 c all formed on a semiconductor substrate. The carrier-travelregion may be connected to the plurality of carrier-storage bins bycarrier-transport channels 1-907. Only three carrier-storage bins areshown, but there may be more. There may be a read-out channel 1-910connected to the carrier-storage bins. Thephoton-absorption/carrier-generation region 1-902, carrier-travel region1-906, carrier-storage bins 1-908 a, 1-908 b, 1-908 c, and read-outchannel 1-910 may be formed by doping the semiconductor locally and/orforming adjacent insulating regions to provide photodetection capabilityand confine carriers. A time-binning photodetector 1-900 may alsoinclude a plurality of electrodes 1-920, 1-922, 1-932, 1-934, 1-936,1-940 formed on the substrate that are configured to generate electricfields in the device for transporting carriers through the device.

In operation, fluorescent photons may be received at thephoton-absorption/carrier-generation region 1-902 at different times andgenerate carriers. For example, at approximately time t₁ threefluorescent photons may generate three carrier electrons in a depletionregion of the photon-absorption/carrier-generation region 1-902. Anelectric field in the device (due to doping and/or an externally appliedbias to electrodes 1-920 and 1-922, and optionally or alternatively to1-932, 1-934, 1-936) may move the carriers to the carrier-travel region1-906. In the carrier-travel region, distance of travel translates to atime after excitation of the fluorescent molecules. At a later time t₅,another fluorescent photon may be received in thephoton-absorption/carrier-generation region 1-902 and generate anadditional carrier. At this time, the first three carriers have traveledto a position in the carrier-travel region 1-906 adjacent to the secondstorage bin 1-908 b. At a later time t₇, an electrical bias may beapplied between electrodes 1-932, 1-934, 1-936 and electrode 1-940 tolaterally transport carriers from the carrier-travel region 1-906 to thestorage bins. The first three carriers may then be transported to andretained in the first bin 1-908 a and the later-generated carrier may betransported to and retained in the third bin 1-908 c. In someimplementations, the time intervals corresponding to each storage binare at the sub-nanosecond time scale, though longer time scales may beused in some embodiments (e.g., in embodiments where fluorophores havelonger decay times).

The process of generating and time-binning carriers after an excitationevent (e.g., excitation pulse from a pulsed optical source) may occuronce after a single excitation pulse or be repeated multiple times aftermultiple excitation pulses during a single charge-accumulation cycle forthe photodetector 1-900. After charge accumulation is complete, carriersmay be read out of the storage bins via the read-out channel 1-910. Forexample, an appropriate biasing sequence may be applied to at leastelectrode 1-940 and a downstream electrode (not shown) to removecarriers from the storage bins 1-908 a, 1-908 b, 1-908 c.

After a number of excitation events, the accumulated signal in eachelectron-storage bin may be read out to provide a histogram havingcorresponding bins that represent the fluorescent emission decay rate,for example. Such a process is illustrated in FIG. 1-10A and FIG. 1-10B.The histogram's bins may indicate a number of photons detected duringeach time interval after excitation of the fluorophore(s) in a reactionchamber. In some embodiments, signals for the bins will be accumulatedfollowing a large number of excitation pulses, as depicted in FIG.1-10A. The excitation pulses may occur at times t_(e1), t_(e2), t_(e3),. . . t_(eN) which are separated by the pulse interval time T. There maybe between 10⁵ and 10⁷ excitation pulses applied to the reaction chamberduring an accumulation of signals in the electron-storage bins. In someembodiments, one bin (bin 0) may be configured to detect an amplitude ofexcitation energy delivered with each optical pulse, and be used as areference signal (e.g., to normalize data).

In some implementations, only a single photon on average may be emittedfrom a fluorophore following an excitation event, as depicted in FIG.1-10A. After a first excitation event at time t_(e1), the emitted photonat time t_(f1) may occur within a first time interval, so that theresulting electron signal is accumulated in the first electron-storagebin (contributes to bin 1). In a subsequent excitation event at timet_(e2), the emitted photon at time t_(f2) may occur within a second timeinterval, so that the resulting electron signal contributes to bin 2.

After a large number of excitation events and signal accumulations, theelectron-storage bins of the time-binning photodetector 1-322 may beread out to provide a multi-valued signal (e.g., a histogram of two ormore values, an N-dimensional vector, etc.) for a reaction chamber. Thesignal values for each bin may depend upon the decay rate of thefluorophore. For example and referring again to FIG. 1-8, a fluorophorehaving a decay curve B will have a higher ratio of signal in bin 1 tobin 2 than a fluorophore having a decay curve A. The values from thebins may be analyzed and compared against calibration values, and/oreach other, to determine the particular fluorophore, which in turnidentifies the nucleotide or nucleotide analog (or any other molecule orspecimen of interest) linked to the fluorophore when in the reactionchamber.

To further aid in understanding the signal analysis, the accumulated,multi-bin values may be plotted as a histogram, as depicted in FIG.1-10B for example, or may be recorded as a vector or location inN-dimensional space. Calibration runs may be performed separately toacquire calibration values for the multi-valued signals (e.g.,calibration histograms) for four different fluorophores linked to thefour nucleotides or nucleotide analogs. As an example, the calibrationhistograms may appear as depicted in FIG. 1-11A (fluorescent labelassociated with the T nucleotide), FIG. 1-11B (fluorescent labelassociated with the A nucleotide), FIG. 1-11C (fluorescent labelassociated with the C nucleotide), and FIG. 1-11D (fluorescent labelassociated with the G nucleotide). A comparison of the measuredmulti-valued signal (corresponding to the histogram of FIG. 1-10B) tothe calibration multi-valued signals may determine the identity “T”(FIG. 1-11A) of the nucleotide or nucleotide analog being incorporatedinto the growing strand of DNA.

In some implementations, fluorescent intensity may be used additionallyor alternatively to distinguish between different fluorophores. Forexample, some fluorophores may emit at significantly differentintensities or have a significant difference in their probabilities ofexcitation (e.g., at least a difference of about 35%) even though theirdecay rates may be similar. By referencing binned signals (bins 1-3) tomeasured excitation energy bin 0, it may be possible to distinguishdifferent fluorophores based on intensity levels.

In some embodiments, different numbers of fluorophores of the same typemay be linked to different nucleotides or nucleotide analogs, so thatthe nucleotides may be identified based on fluorophore intensity. Forexample, two fluorophores may be linked to a first nucleotide (e.g.,“C”) or nucleotide analog and four or more fluorophores may be linked toa second nucleotide (e.g., “T”) or nucleotide analog. Because of thedifferent numbers of fluorophores, there may be different excitation andfluorophore emission probabilities associated with the differentnucleotides. For example, there may be more emission events for the “T”nucleotide or nucleotide analog during a signal accumulation interval,so that the apparent intensity of the bins is significantly higher thanfor the “C” nucleotide or nucleotide analog.

The inventors have recognized and appreciated that distinguishingnucleotides or any other biological or chemical specimens based onfluorophore decay rates and/or fluorophore intensities enables asimplification of the optical excitation and detection systems in ananalytical instrument 1-100. For example, optical excitation may beperformed with a single-wavelength source (e.g., a source producing onecharacteristic wavelength rather than multiple sources or a sourceoperating at multiple different characteristic wavelengths).Additionally, wavelength discriminating optics and filters may not beneeded in the detection system. Also, a single photodetector may be usedfor each reaction chamber to detect emission from differentfluorophores.

The phrase “characteristic wavelength” or “wavelength” is used to referto a central or predominant wavelength within a limited bandwidth ofradiation (e.g., a central or peak wavelength within a 20 nm bandwidthoutput by a pulsed optical source). In some cases, “characteristicwavelength” or “wavelength” may be used to refer to a peak wavelengthwithin a total bandwidth of radiation output by a source.

The inventors have recognized and appreciated that fluorophores havingemission wavelengths in a range between about 560 nm and about 900 nmcan provide adequate amounts of fluorescence to be detected by atime-binning photodetector (which may be fabricated on a silicon waferusing CMOS processes). These fluorophores can be linked to biologicalmolecules of interest such as nucleotides or nucleotide analogs.Fluorescent emission in this wavelength range may be detected withhigher responsivity in a silicon-based photodetector than fluorescenceat longer wavelengths. Additionally, fluorophores and associated linkersin this wavelength range may not interfere with incorporation of thenucleotides or nucleotide analogs into growing strands of DNA. Theinventors have also recognized and appreciated that fluorophores havingemission wavelengths in a range between about 560 nm and about 660 nmmay be optically excited with a single-wavelength source. An examplefluorophore in this range is Alexa Fluor 647, available from ThermoFisher Scientific Inc. of Waltham, Mass. The inventors have alsorecognized and appreciated that excitation energy at shorter wavelengths(e.g., between about 500 nm and about 650 nm) may be required from apulsed laser to excite fluorophores that emit a wavelengths betweenabout 560 nm and about 900 nm. In some embodiments, the time-binningphotodetectors may efficiently detect longer-wavelength emission fromthe samples, e.g., by incorporating other materials, such as Ge, intothe photodetectors active region.

The inventors have also recognized and appreciated that optical pulsesfrom a pulsed laser should extinguish quickly for the detection schemesdescribed above, so that the excitation energy does not overwhelm orinterfere with the subsequently detected fluorescent signal. In someembodiments and referring again to FIG. 1-5, there may be no wavelengthfilters between the waveguide 1-315 and the time-binning photodetector1-322. To avoid interference of the excitation energy with subsequentsignal collection, the excitation pulse may need to reduce in intensityby at least 50 dB within about 100 ps from the peak of the excitationpulse. In some implementations, the excitation pulse may need to reducein intensity by at least 80 dB within about 100 ps from the peak of theexcitation pulse. The inventors have recognized and appreciated thatmode-locked lasers can provide such rapid turn-off characteristics. Insome cases, where emission wavelengths are significantly longer than theexcitation wavelength, simple optical filters may be incorporated overthe photodetectors to further reduce the impact of the excitation pulseon the time-binning photodetectors. According to some embodiments, areduction in intensity of the excitation energy between pulses may bereduced additionally by 20 dB or more if the excitation energy isdirected away from the detection apparatus for the fluorescent signal.For example, the excitation energy may be delivered in a waveguide, asdepicted in FIG. 1-3, propagating in a different direction from thefluorescent-detection path (e.g., the directions of the two paths may beapproximately orthogonal as depicted in the drawing). Reductions inexcitation energy between pulses can also be achieved through waveguidematerial development and device fabrication (e.g., waveguide materialthat exhibits reduced scattering loss and reduced fluorescence and anetching process that produces smooth waveguide sidewalls). Further,scatter of excitation energy off of the reaction chamber may be reducedby choice of chamber geometry, materials, and geometries of surroundingstructures based on results from electromagnetic simulations.

The inventors have also recognized and appreciated that a pulsed lasershould provide enough energy per pulse to excite at least onefluorophore in each of the reaction chambers on the bio-optoelectronicchip for each excitation pulse. For a chip that includes about 65,000reaction chambers and accounting for optical losses throughout thesystem, the inventors have determined that a pulsed laser should provideabout 300 mW or more of average optical power at the excitationwavelength.

The inventors have further recognized and appreciated that a beamquality of the pulsed laser should be high (e.g., an M² value less than1.5), so that efficient coupling can be achieved to an optical couplerand waveguides of a bio-optoelectronic chip 1-140.

A pulsed laser system having the foregoing characteristics and operablein a compact package (e.g., occupying a volume less than about 0.5 ft³)would be useful for portable analytic instruments 1-100, such as aninstrument configured to sequence DNA as described above.

II. Pulsed Laser Embodiments II. A. Mode-Locked Lasers

The inventors have conceived and built a pulsed laser system 1-110 thatachieves the above-described performance specifications in terms ofaverage power, compactness, beam quality, pulse repetition rate,operating wavelength, and turn-off speed of optical pulses. According tosome embodiments, a pulsed laser comprises a solid-state, mode-lockedlaser as depicted in FIG. 2-1A. Optical components of the lasing systemmay be mounted on a base plate 2-105 that measures between about 20 cmand about 40 cm in length, between about 10 cm and about 30 cm inheight, and has a thickness between about 10 mm and about 18 mm. In someimplementations, dimensions of the base plate may be about 30 cm inlength, about 18 cm in height, and about 12 mm in thickness. In someembodiments, 12 mm-diameter optical components (or smaller) may be usedin the laser system and partially recessed into the base plate (asdescribed later in connection with FIG. 2-2A), so that an overallthickness of the lasing system, including optical components andassociated optical mounts may be between 4 cm and about 6 cm. Accordingto some embodiments, a volume occupied by the lasing system may be about30 cm×18 cm×5 cm or about 0.1 ft³.

A pulsed laser may comprise an output coupler 1-111 at an output end ofthe laser cavity, a gain medium 1-105, and a saturable absorber mirror(SAM) 1-119 at an opposite end of the laser cavity. There may bemultiple mirrors within the laser cavity to fold the optical axis 1-125and extend the length of the laser cavity to achieve a desired pulserepetition rate. There may also be beam-shaping optics (e.g. lensesand/or curved mirrors) within the laser cavity to alter a size and/orshape of the intracavity laser beam.

According to some embodiments, the output coupler 1-111 may be ahigh-quality laser optic having a surface quality of 10-5 (scratch anddig) and a wavefront error of at most λ/10. One surface of the outputcoupler may be coated with a multi-layer dielectric to provide areflectance between about 75% and about 90% for the lasing wavelengthλ₁. A second surface of the output coupler may be coated with anantireflection coating, and may be oriented at an angle with respect tothe reflective surface. The coatings on the output coupler may bedichroic, so as to transmit with negligible reflection a pump wavelengthλ_(p) from a diode pump laser that may be used to excite the gain medium1-105. The output coupler may be mounted in a two-axis adjustable mountthat provides angular adjustment with respect to the incident opticalaxis 1-125 about two orthogonal axes. In some embodiments, the outputcoupler may be mounted on a non-adjustable mount.

The gain medium 1-105 may comprise a neodymium-doped material that ismounted in a thermally-conductive mount (e.g., a copper block) whichdissipates heat into the base plate 2-105. To improve heat transfer fromthe gain medium to the copper block, the gain medium may be wrapped inindium foil or any other suitable material that improves heat transferto the thermally-conductive mount. In some cases, the gain medium andthermally-conductive mount may be mounted on a thermo-electric cooler(TEC), which may sink heat into the base plate 2-105. The TEC mayprovide temperature control of the gain medium. In some implementations,the gain medium may comprise neodymium vanadate (e.g., Nd³⁺:YVO₄) havinga length between about 3 mm and about 10 mm. The neodymium dopant levelmay be between about 0.10% and about 1%. End facets of the crystal maybe anti-reflection coated for the lasing wavelength λ₁, which may beabout 1064 nm for neodymium vanadate. The gain medium 1-105 may bemounted in a non-adjustable mount (a mount that provides no fine angularor positional adjustment) in an orientation where end facets of the gainmedium have normal vectors oriented at an angle between about 1 degreeand about 3 degrees to the optical axis 1-125 of the laser cavity.

The saturable absorber mirror 1-119 may comprise a multilayersemiconductor structure (e.g., a multiple quantum well) and a highreflector. The semiconductor structure may exhibit nonlinear opticalabsorption. For example, the SAM may exhibit higher absorption at lowoptical intensities, and may bleach or exhibit little absorption at highoptical intensities. The semiconductor structure may be spaced from thehigh reflector in the SAM so that the semiconductor structure is locatedat approximately a peak intensity of an optical standing wave created bythe optical field incident on and reflected from the high reflector. Anexample of a SAM is part number SAM-1064-5-10ps-x available from BATOPOptoelectronics GmbH of Jena, Germany. Because of the SAM's nonlinearoptical absorption, the laser preferentially operates in a pulsed modeof operation (passively mode locked). In some implementations, a SAM maybe mounted on a rotating and/or transverse-positioning mount, so thatthe SAM's surface may be moved in a direction transverse to the opticalaxis 1-125. Should the SAM become damaged, the SAM may be moved and/orrotated so that the intracavity beam is focused onto an undamaged regionof the SAM. In other embodiments, the SAM may be mounted on anon-adjustable mount.

To excite the gain medium 1-105, a continuous-wave output (indicated bythe black dotted line in FIG. 2-1A) from a laser diode in a pump module2-140 may be focused into the gain medium using a coupling lens 2-142.In some embodiments, a beam from the laser diode may have a rectangularor square cross section and may diverge slightly (e.g., between about 5degrees and about 10 degrees). In some implementations, a focal lengthof the coupling lens 2-142 may be between about 20 mm and about 30 mm.Unabsorbed pump radiation may pass through a laser-cavity turning mirror2-115 and be absorbed in a beam dump 2-116.

Other excitation sources may be used to pump the gain medium 1-105 inother embodiments, and the invention is not limited to laser diodes. Insome embodiments, a fiber or fiber-coupled laser may be used to pump thegain medium 1-105 of the pulsed laser 1-110. A fiber laser may comprisean active optical fiber as part of the fiber-laser cavity that is pumpedby one or more laser diodes. A fiber-coupled laser may comprise one ormore laser diodes having their outputs coupled into an optical fiber. Anoutput beam from a fiber carrying optical energy from the fiber laser orfiber-coupled laser may be directed to and focused into the gain mediumusing the same or similar optics that are used for a laser diode. Anoptical beam from a fiber may have a more circular, homogenous, and/orGaussian (or top-hat-shaped) spatial profile than a beam directly from ahigh-power laser diode pump source. The pump source may or may not bemounted on a fixture other than base plate 2-105 in some embodiments,and an end of the fiber carrying pump energy may be attached to a mounton the pulsed laser that is located on the same side or opposite side ofthe base plate as the gain medium 1-105, or may be mounted remotely fromthe laser cavity structure.

The focal length of coupling lens 2-142, the size of the pump beam, andthe lens' distance from the gain medium 1-105 determine the size(cross-section dimensions) of the pump beam in the gain medium. Inembodiments, the size of the pump beam in the gain medium isapproximately matched (e.g., to within 15%) to a mode-field size of thelaser beam in the gain medium. The mode-field size of the laser beam inthe gain medium may be determined predominantly by a focal length of acurved mirror 2-117 within the laser cavity, the waist of the pump beamin the gain medium, and a distance of the gain medium from the curvedmirror. In some embodiments, a focal length of a curved mirror 2-117 maybe between about 200 mm and about 300 mm.

According to some embodiments, the position of the pump beam in the gainmedium 1-105 is adjusted in two degrees of freedom (in directionstransverse to the optical axis 1-125 of the laser cavity) by adjustablemounts in the pump module 2-140. These adjustable mounts are outside thepulsed laser cavity. The adjustable mounts for the pump beam may be usedsteer the pump beam to overlap the laser beam in the gain medium 1-105and improve pumping efficiency of the laser.

To utilize the nonlinear optical absorption in the SAM 1-119, a focusinglens 2-123 is incorporated into the laser cavity near the SAM. Accordingto some embodiments, a focal length of the focusing lens 2-123 isbetween about 70 mm and about 130 mm, and the SAM is locatedapproximately at the focal length of the focusing lens 2-123. Thefocusing lens reduces the spot size of the intracavity laser beam on theSAM, boosting its intensity.

The inventors have discovered, somewhat surprisingly, that for somelaser-cavity configurations the spot size of the laser beam on the SAMis more sensitive to changes in distance between the curved mirror 2-117and the laser's output coupler 1-111 than to changes in distance betweenthe focusing lens 2-123 and SAM 1-119. This result relates to theextended cavity length between the curved mirror 2-117 and the focusinglens 2-123. The extended cavity length comprises multiplehigh-reflective optics 2-121 (e.g., having reflectivities between about99.9% and about 99.999%) that bounce the optical pulses back and forthon the base plate 2-105, increasing the travel distance between thecurved mirror 2-117 and focusing lens 2-123. Along this extended cavitylength, the laser beam may be approximately collimated. Changes in thedistance between the curved mirror 2-117 and output coupler 1-111 canaffect collimation in the extended cavity, and the increased cavitylength amplifies changes in beam size at the focusing lens 2-123. Thisamplification in turn affects the spot size in the SAM more stronglythan changes in distance between the focusing lens 2-123 and SAM 1-119.

In some embodiments, fine positional control (e.g., a micro-positioningstage) may be included with the output coupler 1-111 and/or curvedmirror 2-117 to provide operational tuning of the distance between theoutput coupler and curved mirror. Because the focal length of the curvedmirror 2-117 may have a specified tolerance (e.g., ±2 mm), a range ofthe fine positional control may extend over at least a range thatincludes the specified focal length tolerance of the curved mirror. Insome implementations, fine positional control may not be included withthe output coupler 1-111 and/or curved mirror 2-117. Instead, the focallength of the curved mirror may be determined prior to installation, andthe curved mirror located in the cavity accordingly. In some cases, theoutput coupler 1-111 may be mounted on a non-adjustable mount, and thecurved mirror 2-117 may be mounted on a two-axis tilt-adjustment mount.In some embodiments, the adjustable mount for the curved mirror may bethe only adjustable mount in the pulsed laser cavity that can beadjusted while the laser is operating and provide two degrees of freedomin adjusting the laser beam. Therefore, the pulsed laser may only haveoperational adjustment only over two degrees of freedom via the curvedmirror mount located between the cavity end mirrors. The remainingoptical components of the laser cavity depicted in FIG. 2-1 may bemounted on non-adjustable mounts. Using non-adjustable mounts and onlyone adjustable mount can make the pulsed laser more reliable and robustduring operation, and reduce drift and misalignment of opticalcomponents in the pulsed laser.

Additional elements may be included in the laser cavity in someembodiments. For example, an intracavity beam-steering module 2-130 maybe included before and/or after the focusing lens 2-123 (depicted beforethe focusing lens in FIG. 2-1A). The intracavity beam-steering modulemay comprise anti-reflection coated optical flats that can be angledwith respect to the laser beam about two orthogonal axes to translatethe laser beam in two directions. When optical flats for an intracavitybeam-steering module 2-130 are located before the focusing lens 2-123,translation of the laser beam will result in predominantly a change ofincident angle of the laser beam on the SAM 1-119. For optical flatslocated after the focusing lens, translation of the laser beam willresult predominantly in a change in position of the laser beam on theSAM. In some implementations, an intracavity beam-steering module 2-130may be used to provide automated, fine tuning of cavity alignment (e.g.,automated tuning and/or alignment based on feedback signals derived fromaverage power of the laser or other pulsed-operation characteristics).In some cases, an intracavity beam-steering module may be used toreposition the laser beam on the SAM (e.g., moving the laser beam shouldthe SAM become damaged at a focal spot).

According to some embodiments, rather than using rotating optical flatsfor intracavity re-alignment of the laser beam, another possibility isto induce asymmetric thermal gradients in the gain medium 1-105 that canaffect thermal lensing within the gain medium. Asymmetric thermalgradients in the gain medium 1-105 can cause small angular deflectionsin the intracavity laser beam as it passes through the gain medium. Insome implementations, one or more temperature-controlling devices (e.g.,resistive heating elements, TEC coolers, or a combination thereof) maybe coupled to one or more sides of the gain medium. According to someembodiments, the gain medium 1-105 may have four independently-operableheating elements thermally coupled to four faces (four longitudinaledges) of the gain medium. Thermal coupling may comprise thermal epoxyor indium foil located between a temperature-controlling device and faceof the gain medium. Each temperature-controlling device may also includethermal coupling to a heat sink (such as the laser block) on an oppositeside of the temperature-controlling device. In some cases, one or moreof a first pair of temperature-controlling devices located on firstopposing faces of the gain medium may provide beam deflection indirections normal to the two first opposing faces (e.g., ±x directions).One or more of a second pair of temperature-controlling devices locatedon an orthogonal pair of second opposing faces of the gain medium mayprovide beam deflection in orthogonal directions (e.g., ±y directions)By selectively altering temperatures at the temperature-controllingdevices, the intracavity laser beam may be steered and re-aligned. Thesteering and re-alignment may change the position of the intracavitybeam on the SAM 1-119. In some cases, the curved mirror 2-117 or acavity end mirror may be additionally adjusted to re-align theintracavity laser beam.

In some embodiments, a pulsed laser 1-110 may provide adjustable mountsfor one or a few of the optical components within the laser. Anadjustable mount may allow an operator to finely adjust the positionand/or orientation of the optical component while the laser is lasing,so that operation of the laser can be tuned for stability, beam quality,output power, and/or pulse characteristics. Fine tuning may be achievedby micrometers and/or finely-threaded screw adjustments on mirrormounts, for example. In some embodiments, a pulsed laser 1-110 mayinclude adjustable mounts only for one or more of the output coupler1-111 (angular adjustments), the curved mirror 2-117 (position andangular adjustments), and the SAM 1-119 (angular adjustments). In someimplementations, the coupling lens 2-142 may include an adjustablepositioning mount. The remaining optical components of the laser cavitymay be aligned during manufacture in fixed, non-adjustable mounts. Anexample of an integrated, self-aligning, non-adjustable mount isdescribed below in connection with FIG. 2-2A.

The inventors have recognized and appreciated that stable, pulsedoperation of the laser 1-110 may occur for a range of relative spotsizes of the intracavity laser beam in the gain medium 1-105 and on theSAM 1-119. For example, a ratio of a minimum beam waist in the gainmedium to a focused beam waist on the SAM may be between about 4:1 andabout 1:2. According to some embodiments, a beam radius (1/e² value ofthe intensity) in the gain medium may be between about 20 μm and about200 μm, and a beam radius (1/e² value of the intensity) on the SAM maybe between about 50 μm and about 200 μm. For ratios and beam radiioutside these ranges, the pulsed operation may become unstable and thelaser may Q-switch, which can damage the SAM. According to someembodiments, a specification of the SAM may be its saturation fluence,and an intensity of the focused laser beam on the SAM may beproportional to the saturation fluence. For example, the intensity ofthe focused laser beam may be between approximately 1 times and 10 timesthe saturation fluence of the SAM.

The inventors have recognized and appreciated that average power and/orspectral characteristics of the pulsed laser may be determinative ofstable, mode-locked operation. For example, if the laser's average powerduring mode-locked operation falls below a certain value, there may notbe enough nonlinear optical absorption in the SAM to support modelocking. The laser may then Q-switch and damage the SAM. In some cases,rapid fluctuations of the laser's average output power may indicate thatthe laser is Q-switching in addition to mode locking, which can damagethe SAM. In some embodiments, a sensor 2-154 (e.g., a photodiode) may beincluded and arranged to sense optical power produced by the laser1-110. If the sensed average laser power drifts below a preset level orpower fluctuations are detected, an automated cavity alignment routinemay be executed to recover power and/or the laser may be shut off forservicing.

As may be appreciated, alignment of the laser-cavity optics may bedifficult because of the high number of mirrors. In some embodiments, apulsed laser may include mounting features 2-118 (e.g., screw holesand/or registration features) located along the optical axis of thelaser cavity, e.g., between the curved mirror 2-117 and focusing lens2-123. The mounting features 2-118 may be configured to receive anoptical mount in which a second output coupler may be mounted. When theoptical mount and second output coupler are in place, the laser may bealigned to lase in continuous-wave mode with a shortened laser cavity.The second output coupler may transmit a small amount of power (e.g., 2%or any other suitable value), and provide a laser beam that can be usedto align optical components of laser between the inserted optical mountand the SAM 1-119. Once these remaining components are aligned, theinserted optical mount may be removed, so that the laser 1-110 can betuned to operate in pulsed mode with the full cavity length.

The inventors have recognized and appreciated that heat from the diodepump module 2-140 can adversely affect operation of the pulsed laser1-110. For example, heat from the diode pump module 2-140 can warm asignificant area of the base plate 2-105 and change alignment oflaser-cavity optics over time. To avoid deleterious effects caused byheat from the diode pump laser, the diode pump module 2-140 may bemounted through a hole 2-145 in the base plate 2-105. According to someembodiments, a beam from the laser diode may be directed (in a directioncoming out of the page) to a dichroic mirror oriented at 45° within thediode pump module 2-140 and that lies on the output beam path 2-125 ofthe pulsed laser. The dichroic mirror may include adjustments that canalign the laser diode's pump beam to the gain medium 1-105 and opticalaxis of the laser cavity.

In some embodiments, the diode pump module 2-140 may attach to the baseplate 2-105 using thermally insulating mounting hardware. For example,nylon screws may be used to attach the diode pump module and nylon orceramic washers may be placed between the base plate and mountingsurfaces of the diode pump module. In some implementations, smallstainless steel screws (e.g., screw sizes of 4-40 or smaller) may beused with nylon or ceramic washers. Additionally, TEC, cooling fins,and/or forced-air cooling of the diode pump module may be implemented ona reverse side of the base plate 2-105 so that heat is conducted awayfrom the base plate and laser-cavity optics. According to someembodiments, the diode pump module 2-140 may be located within about 2cm of an edge of the base plate 2-105, and the dissipated heat directedtoward the edge and away from the base plate by a fan, for example. Thebase plate 2-105 may serve additionally as a wind screen, protecting thelaser optics and laser cavity on one side of the base plate from airflow or turbulence on the reverse side of the plate where heat isremoved. In some implementations, a TEC may be connected to feedback andcontrol circuitry and used to maintain the diode pump laser at a desiredoperating temperature.

An example of a partially-assembled, portable instrument 1-100 thatincludes a pulsed laser 1-110 is shown in FIG. 2-1B. Also visible in thephotograph are a printed circuit board 1-130 on which mounts abio-optoelectronic chip 1-140. A beam-steering module 1-150 may alsoattach to the PCB 1-130. In this embodiment, optics of the pulsed laserare mounted on an optical breadboard having many tapped holes. In someembodiments, some optics for the pulsed laser may mount in integrated,self-aligning, optical mounts formed in the base plate 2-105.

An example of an integrated, self-aligning, optical mount 2-210 isdepicted in FIG. 2-2A. An integrated optical mount 2-210 may comprise anaxial trench 2-220 machined or otherwise formed into the base plate2-105 of a pulsed laser 1-110. The axial trench 2-220 may extend in adirection parallel to an optical axis of the pulsed laser cavity. Anintegrated optical mount may further comprise coplanar surfaces 2-230formed approximately transverse to the axial trench 2-220. The coplanarsurfaces may be formed by machining or milling a short trench in adirection that is approximately orthogonal to the axial trench 2-220. Insome cases, the coplanar surfaces may be oriented at a small angle, sothat back reflections from a mounted optic will be displaced from theoptical axis of the laser cavity. At the base of the axial trench 2-220there may be sloped surfaces 2-240 (only one is visible in FIG. 2-2A).The sloped surfaces 2-240 may be machined, milled, or otherwise formednear the base of the axial trench and located on opposite sides of theaxial trench 2-220. The sloped surfaces may be inclined in a directiontoward the coplanar surfaces 2-230, and provide support for an opticmounted thereon.

An optical component 2-250 for a pulsed laser, for example, may besupported by the integrated optical mount 2-210, as depicted in FIG.2-2B. The optic 2-250 may comprise a cavity mirror, a lens within thelaser cavity, or the gain medium 1-105, for example. In some cases, theoptic 2-250 may be mounted by itself in the integrated optical mount2-210, as depicted in the drawing. In other embodiments, an optic may bemounted within a supporting fixture (e.g., an annular plate, anadjustable mount) that can be placed in the integrated optical mount2-210.

According to some embodiments, an optical component 2-250, or supportingfixture, may include a flat surface that registers to and rests againstthe coplanar surfaces 2-230 of the integrated optical mount 2-210. Theoptic or fixture may be retained in the integrated mount by a compliantretaining device (e.g., an O-ring mounted on a bar that can be fastenedto the base plate, a flexible plastic bar or arm, etc.). The compliantretaining device may contact a top edge of the optic 2-250 or supportingfixture, and may exert forces on the optic or fixture in directionstowards inclined surfaces 2-240 and the coplanar surfaces 2-230. A loweredge of the optic 2-250 or supporting fixture may contact points on theinclined surfaces 2-240. The inclined surfaces 2-240 may also provide aforce against the optic or fixture having a component that is directedin part toward the coplanar surfaces 2-230. The contact points at theinclined surfaces 2-240 and forces directed toward the coplanar surfaces2-230 can self-align the optic or fixture to a desired orientation andlocation within the laser cavity. In some implementations, an optic orsupporting fixture may be bonded in the integrated optical mount (e.g.,with an adhesive) in an aligned orientation.

One or more integrated optical mounts 2-210 may be formed in a baseplate of a pulsed laser 1-110, according to some embodiments. In somecases, an axial trench 2-220 may extend through several integratedoptical mounts, as depicted in FIG. 2-2A. Among the advantageousfeatures of an integrated optical mount are a lowering of the pulsedlaser's optical axis. This can reduce effects of mechanical vibrationsthat might otherwise couple into and be amplified by optical mountsextending from a surface of the base plate, and can reduce effects ofthermal expansion (e.g., slight warping of the base plate 2-105) thatmight otherwise be amplified by motion of optical mounts extending froma surface of the base plate.

Referring again to FIG. 2-1, an output of a pulsed laser 1-110 may befocused through a lens 2-164 into a frequency-doubling crystal 2-170 tohalve the optical wavelength of the output pulses. For example, thepulsed laser 1-110 may produce pulses with a characteristic wavelengthof about 1064 nm, and the frequency-doubling crystal 2-170 may convertthe wavelength to about 532 nm. The frequency-doubled output may be usedto excite fluorophores having different emission characteristics at thebio-optoelectronic chip 1-140.

In some embodiments, a half-wave plate 2-160 may be mounted in arotatable mount with its rotation angle controlled by an actuator 2-162,and may be located in the output optical path of the pulsed laser beforethe frequency-doubling crystal 2-170. According to some embodiments, anactuator 2-162 may comprise a stepper motor, a piezoelectric motor, agalvanometer having precision bearings and configured to rotate anoptical component, a DC motor, or any other suitable actuationmechanism. Rotating the half-wave plate 2-160 can alter the polarizationof the laser's output pulses and change the second-harmonic conversionefficiency in the frequency-doubling crystal 2-170. Control of thehalf-wave plate can then be used to control an amount of power at thefrequency-doubled wavelength that is delivered to the bio-optoelectronicchip 1-140. By rotating the half-wave plate (or the frequency-doublingcrystal), the optical power at the frequency-doubled wavelength can bevaried precisely by small amounts over a large range (e.g., over anorder of magnitude or more), without affecting the operation of thelaser at the fundamental wavelength. That is, the power at thefrequency-doubled wavelength can be altered without affecting themode-locking stability, thermal dissipation, and other characteristicsof the pulsed laser 1-110. In some embodiments, other adjustments may beused additionally or alternatively to control frequency-doubled powerwithout affecting the fundamental laser operation. For example, anincident angle of the pulsed-laser beam on the frequency-doublingcrystal 2-170 and/or distance between the lens 2-164 andfrequency-doubling crystal may be controlled in an automated manner toalter and/or maximize the frequency-doubling efficiency.

In some embodiments, the frequency-doubled output pulses may be directedby a turning mirror 2-180 to a beam steering module 1-150. The turningmirror 2-180 may be dichroic, such that it transmits optical radiationwhich has not been down-converted by the frequency-doubling crystal2-170 to a beam dump (not shown).

In operation, a pulsed laser 1-110 that employs Nd³⁺:YVO₄ as the gainmedium, having a length of 7 mm and a doping level of about 0.25%, canproduce pulses at 1064 nm having a FWHM value of approximately 20 ps.The pulse extinguishes by approximately 80 dB within 100 ps from thepeak of the pulse. The pulse repetition rate is approximately 90 MHz,and the average power of the pulsed laser at the fundamental wavelengthis about 900 mW. The average frequency-doubled power is about 300 mW.The AC power required to operate the laser is less than about 20 Watts.The laser is compact, occupies a volume of less than 0.1 ft³, weighsapproximately 10 pounds, and can be readily incorporated as a moduleinto a portable analytic instrument, such as a table-top instrument forsequencing DNA.

Additional mode-locked laser configurations and features may be used insome implementations. FIG. 3-1 depicts just one example of a compactmode-locked laser 3-100. In overview, a compact mode-locked laser maycomprise a diode pump source 3-105, gain medium 3-107, afrequency-doubling element 3-109, an optical delay element 3-110, andtwo laser cavity end mirrors TC₁ and saturable absorber mirror 3-120.The gain medium 3-107 may be excited by the diode pump source 3-105 at awavelength λ_(p) to produce optical emission at a lasing wavelength λ₁.The frequency-doubling element 3-109 may convert the lasing wavelengthto a frequency-doubled output wavelength at λ₂ that is one-half thelasing wavelength.

According to some embodiments, a pump wavelength λ_(p) for any of thedepicted optically-pumped lasing systems may be between approximately450 nm and approximately 1100 nm. A lasing wavelength λ₁ for any of thedepicted lasing systems may be between approximately 800 nm andapproximately 1500 nm, according to some implementations. In some cases,an output wavelength λ₂ for any of the depicted lasing systems may bebetween approximately 400 nm and approximately 750 nm. In some cases, anoutput wavelength λ₂ may be between approximately 500 nm andapproximately 700 nm. An output pulse duration may be between about 1picosecond and about 100 picoseconds, according to some embodiments. Insome cases, the output pulse duration may be between about 1 picosecondand about 30 picoseconds.

In some embodiments, an optical pump source 3-105, gain medium 3-107,and frequency-doubling element 3-109 for any of the depicted lasingsystems may be selected to produce a desired output wavelength λ₂. Forexample, if a green output wavelength is desired, the gain medium may beNd:YAG, or Nd:YLF, which lase at 1064 nm and 1053 nm, respectively. Thefrequency-doubling element 3-109 may be KTP or BBO in someimplementations, and the pump source may comprise one or more laserdiodes that lase at approximately 800 nm. Other materials may beselected for other desired output wavelengths λ₂. For example,Cr:Forsterite may be used as a gain medium, which may lase at 1280 nmand be frequency doubled to 640 nm (in the red region of the opticalspectrum). In some embodiments, Pr:LiYF₄ may be used as the gain medium3-107 to lase at 640 nm (in the red) directly, without frequencydoubling. The inventors have recognized and appreciated that Nd:YVO₄ maybe used as a gain medium to lase at one or two wavelengths 1064 nmand/or 1342 nm, which may be doubled to 532 nm (green) and/or 671 nm(red). The inventors have also recognized and appreciated thatsum-frequency generation may be performed in a nonlinear crystal toobtain additional wavelengths. For example, pulses at the two lasingwavelengths from Nd:YVO₄ may be mixed in a nonlinear crystal to produceradiation at approximately 594 nm. Additional wavelengths that may beproduced through selection of gain medium, optical pump source, annonlinear element 3-109, and that are of interest for excitingfluorophores include, but are not limited to: 515 nm, 563 nm, 612 nm,632 nm, and 647 nm. Different gain media include, but are not limitedto: neodymium-doped yttrium aluminum garnet (Nd:YAG), ytterbium-dopedYAG (Yb:YAG), ytterbium-doped glass (Yb:glass), erbium-doped YAG(Er:YAG), or titanium-doped sapphire (Ti:sapphire).

In some implementations, a compact, diode-pumped, mode-locked laser maycomprise a modified, high-power, laser pointer. High-power laserpointers are available at moderate cost, and the inventors haverecognized and appreciated that such a laser pointer may be modified tocreate a compact, mode-locked laser. For example, a dichroic mirror DC₁may be inserted between a diode pump source 3-105 and the laser gainmedium 3-107. The dichroic mirror may replace an end mirror of the lasercavity, so that the cavity length can be increased to incorporateadditional optical components. The dichroic mirror DC₁ may reflectsubstantially all of the lasing wavelength λ₁, and transmitsubstantially all of the pump wavelength λ_(p).

A dichroic mirror DC₁ may allow a beam from the laser cavity to bedirected to the optical delay element 3-110. An output from the opticaldelay element may be sent to a saturable absorber mirror 3-120. Thesaturable absorber mirror 3-120 may be added to provide anintensity-dependent loss element in the laser cavity that will mode-lockthe laser pointer and produce ultrafast optical pulses.

According to some embodiments, a diode pump source 3-105 provides anoptical pump beam at a wavelength λ_(p) that is operated on by one ormore lenses of an optical system OS₁ and directed to the gain medium3-107. The pump wavelength may be between approximately 700 nm andapproximately 900 nm, according to some embodiments. An example of alaser diode pump source is laser diode model FL-FM01-10-808 availablefrom FocusLight Coroporation of Xi'an, Shaanxi, China. In someembodiments, the diode pump source 3-105 may be thermally cooled todissipate heat generated by the pump source. For example, a thermalelectric cooler (TEC) may be thermally coupled to the diode pump sourceto extract heat from the diode assembly. In some implementations, thegain medium 3-107 and/or the frequency doubling element 3-109 may alsobe temperature controlled, for example, using one or more thermalelectric coolers 3-103.

In some implementations, TECs may not be used. Instead, opticalcomponents that may experience elevated heat levels (e.g., diode pumpsource, gain medium, nonlinear optical elements) may be mounted onthermally conductive sinks that can conduct and/or dissipate heat fromthe optical component. In some embodiments, thermal sinks may comprisesolid copper mounts that are in thermal contact with an opticalcomponent and with a thermally-conductive and/or dissipative supportplate. In some cases, a thermally conductive film (e.g., a malleableindium film) may be placed between a thermal sink and optical componentto improve heat conduction from the component to the mount.

A mode-locked laser may further comprise a first optical system OS₁ thatis configured to reshape and/or change the divergence of the beam fromthe pump source 3-105. For example, the first optical system OS₁ mayincrease or decrease the size of the beam from the pump source, so thatthe pump source beam waist will approximately match a beam waist of thelaser beam at the gain medium. Additionally or alternatively, the firstoptical system may change the cross-sectional shape of the beam, forexample, from elliptical to circular or to a square shaped beam. In someembodiments, the inventors have found that a square- orrectangular-shaped beam from a diode pump source 3-105 is desired forpumping the gain medium 3-107, and may markedly improve the pumpingefficiency of mode-locked laser.

The first optical system OS₁ may comprise one or more cylindricallenses, in some embodiments. For example, the first optical system maycomprise a pair of crossed cylindrical lenses. The first cylindricallens may have a short focal length (e.g., less than about 5 mm) and thesecond cylindrical lens may have a longer focal length. In someimplementations, the first cylindrical lens may comprise a length ofoptical fiber having a diameter less than about 150 microns. It's focallength may be less than 500 microns. The second cylindrical lens mayhave a focal length that is between about 5 mm and about 10 mm.

In some embodiments, a mode-locked laser cavity may comprise a pluralityof optical components as depicted in FIG. 3-1. One end of the lasercavity may comprise a trichroic mirror TC₁ in some embodiments. Thetrichromatic mirror may have a multilayer coating that is designed toreflect the lasing wavelength λ₁ and the pump wavelength λ_(p), and topass the frequency-doubled output wavelength λ₂. The laser cavity mayfurther include a second optical system OS₂ that is configured toreshape and/or change the divergence of the beam from the pump sourceand laser beam into the gain medium 3-107 and nonlinear optical element3-109. In some embodiments, there may be a fifth optical system (notshown) located between the gain medium and nonlinear optical element.The laser cavity may include the dichroic reflector DC₁, describedabove, that reflects the intracavity laser beam to the optical delayelement 3-110. The optical delay element may be configured to addoptical path length to the laser cavity in a compact configuration. Forexample, the optical delay element 3-110 may comprise an optical systemthat measures less than 5 cm on each side and yet provides an opticalpath length within the element that is greater than about 40 cm inlength. In some embodiments, an optical delay element may add an amountof optical path length to a laser cavity that is greater than anytransverse dimension of a base structure or housing on or in which thelaser cavity is disposed. The laser cavity may further include a thirdoptical system OS₃, comprising one or more lenses, that is configured toreshape and/or focus the beam from the optical delay element onto thesaturable absorber mirror 3-120. A laser beam 3-101 within the lasercavity may reflect back-and-forth between the trichroic mirror TC₁ andthe saturable absorber mirror 3-120.

According to some embodiments, the mode-locked laser 3-100 may furtherinclude an output optical system OS₄ and an optical filter F₁. Theoutput optical system may be configured to reshape and/or change thedivergence of the output beam from the laser cavity. The filter may beconfigured to absorb or block one or both of the pump wavelength λ_(p)and the lasing wavelength λ₁.

In operation, the pump beam from the diode pump source may be reshapedwith the optical system OS₁ to efficiently excite the gain medium 3-107.The saturable absorber mirror 3-120 (an example of which is described infurther detail below) exhibits an intensity-dependent loss, such thatlow intensities are absorbed by the mirror and high intensities arereflected by the mirror with a low loss. Because of the mirrorsintensity-dependent loss, the laser preferentially operates in amode-locked state with short, high-intensity pulses. In this state,high-intensity pulses are reflected from the saturable absorber mirror3-120 with low loss. In pulsed operation, the pulses circulateback-and-forth in the laser cavity between the two end mirrors TC₁,3-120, and are frequency doubled by the frequency-doubling element3-109. In this manner, the mode-locked laser produces a train of outputpulses at a doubled wavelength λ₂.

Examples of optical delay elements 3-110 are depicted in FIG. 3-2Athrough FIG. 3-2D. According to just one embodiment, an optical delayelement may comprise an argyle block, as depicted in the plan view ofFIG. 3-2A. The argyle block may comprise a first right-angle prism 3-112and a second right-angle prism 3-114. According to some embodiments, theperpendicular side faces of the prisms may be uncoated, though in otherembodiments the perpendicular faces may include high-reflectivecoatings. In some implementations, a length of a perpendicular face onone of the prisms may measure between about 20 mm and about 60 mm. Eachprism may be formed of any suitable optical quality glass, for exampleBK-7 or fused silica. For high thermal stability, the delay element maybe formed from an ultra-low expansion glass such as ULE, available fromCorning. The side faces of the prisms may be polished to be of highoptical quality, for example, having a flatness of λ/10 or better.

The first prism 3-112 and second prism 3-114 may be offset and adheredtogether, as depicted in the drawing. The prisms may be adhered viaoptical bonding or using an optical adhesive. In some implementations,the optical delay element 3-110 may be formed from a single piece ofglass by cutting and polishing. The laser cavity beam 3-101 may enterthrough a first port of the delay element and be reflected internallyalong a circuitous optical path, depicted as the dotted line, beforeexiting a second port of the argyle block. According to someimplementations, the delay element is double-passed to double theoptical path length in the element within the laser cavity.

Another embodiment of an optical delay element 3-212 is depicted in FIG.3-2B. According to some embodiments, the optical delay element maycomprise a single optical block that is formed in a rectangular shape.The delay element 3-212 may comprise perpendicular edge faces 3-230 thatreflect a laser beam back-and-forth within the delay element, asdepicted in the drawing by the dotted line. The delay element mayfurther include two polished faces that provide an entry port 3-232 andexit port 2-234 for the delay element. The perpendicular side faces maybe uncoated in some embodiments, or coated with a high-reflectivecoatings (e.g., multilayer coatings) in other embodiments. The delayelement 3-212 may be doubled-passed to increase the optical path lengthwithin the laser cavity. In some implementations, a maximum length of anedge of the delay element may measure between about 20 mm and about 60mm. The thickness of the block, measured in a direction into the page,may be between about 5 mm and about 20 mm. The delay element 3-212 maybe formed of any suitable optical quality glass, as described above. Thereflective edge faces may be polished to be of high optical quality, forexample, having a flatness of λ/10 or better.

FIG. 3-2C depicts yet another embodiment of an optical delay element3-214. According to some embodiments, the delay element may comprise apair of planar mirrors M₁, M₂ that are spaced a distance D apart attheir centers and inclined at a slight angle α with respect to eachother. Each mirror may have a length L. The spacing of the mirrors D maybe between about 10 mm and about 50 mm, according to some embodiments.The length of the mirrors L may be between about 20 mm and about 60 mm,according to some embodiments. The angle α may be between about 0° andabout 10°, according to some embodiments. The height of the mirrors M₁,M₂, measured along a direction into the page, may be between about 5 mmand about 20 mm. The mirrors M₁, M₂ may be formed of any suitableoptical quality glass, as described above. The reflective surfaces ofthe mirrors may be polished to be of high optical quality, for example,having a flatness of λ/10 or better. The reflective surfaces may becoated with high-quality, high-reflective, multilayer coatings and havea reflectivity greater than about 99.5% in some implementations. In someembodiments, the reflectivities may be greater than about 99.9%. In someembodiments, the reflectivities may be greater than about 99.99%. Insome implementations, the reflectivities may be greater than about99.999%.

Another embodiment of an optical delay element 3-216 is depicted in FIG.3-2D. This embodiment may comprise a solid block analog to theembodiment depicted in FIG. 3-2C. According to some implementations, anoptical delay element 3-216 may comprise a solid block of opticalmaterial having five surfaces as depicted in the drawing. Two surfaces3-234 may be inclined at a slight angle α with respect to each other.The surfaces may include high reflective coatings to reflect an opticalbeam 3-101 back-and-forth between the surfaces along a dotted path asindicated in the drawing. The delay element 3-216 may further includetwo uncoated or anti-reflection coated surfaces 3-232 that provide anentry port and exit port to and from the delay element. According tosome embodiments, the delay element may be arranged so that theintra-cavity laser beam 3-101 enters and exits the delay element atBrewster's angle. The delay element 3-216 may be formed of any suitableoptical quality glass, as described above. The reflective surfaces 3-234may be polished to be of high optical quality, for example, having aflatness of λ/10 or better. The reflective surfaces may be coated withhigh-quality, high-reflective, multilayer coatings and have areflectivity greater than about 99.5% in some implementations. In someembodiments, the reflectivities may be greater than about 99.9%. In someembodiments, the reflectivities may be greater than about 99.99%. Insome implementations, the reflectivities may be greater than about99.999%.

An advantage of solid-block delay elements 3-110, 3-212, 3-216 depictedin FIG. 3-2A, FIG. 3-2B and FIG. 3-2D is that these elements do notrequire as precise alignment when inserted into the laser cavity asmulti-component delay elements such as two mirrors of FIG. 3-2C.However, solid block components will require more care during amanufacturing process which may lead to increased manufacturing cost.The multi-component delay element 3-214 depicted in FIG. 3-2C will notrequire as much care during a manufacturing process, however it willrequire more care and more precise alignment of the mirrors with respectto each other when added to a laser cavity.

Other mode-locked laser designs incorporating optical delay elements maybe implemented in a compact, ultrashort pulsed laser system. FIG. 3-3Athrough FIG. 3-3C depict additional embodiments of compact, ultrafastmode-locked lasers. FIG. 3-3A depicts an embodiment of a saturableabsorber mirror (SAM) mode-locked laser 3-300 for which the frequencydoubling element 3-109 is located outside the laser cavity. Elements ofthe mode-locked laser that are similar to elements of the mode-lockedlaser 3-100 described in connection with FIG. 3-1 are numbered withsimilar reference numbers and their description is not repeated.According to some embodiments, a SAM mode-locked laser may include anoutput coupler TC₁ and a saturable absorber mirror 3-120 as cavity endmirrors. The output coupler may comprise a trichroic mirror that isconfigured to pass the pump wavelength λ_(p) and be highly reflective tothe lasing wavelength λ₁ and the frequency doubled wavelength λ₂. Insome embodiments, the output coupler TC₁ may transmit between about 2%and about 15% of the lasing wavelength λ₁. A dichroic mirror DC₂ may belocated in the laser cavity to reflect the pump wavelength λ_(p) backthrough the gain medium 3-107 and to transmit the lasing wavelength λ₁to the delay element 3-110. The output beam from the laser cavity may bedirected to the frequency doubling element 3-109 that may be locatedoutside the laser cavity. A filter F₁ may be included to block thelasing wavelength, and optionally the pump wavelength.

FIG. 3-3B depicts an embodiment of a nonlinear mirror mode-locked (NMM)laser 3-302, according to some embodiments. This embodiment may, or maynot, use a saturable absorber. Instead, a frequency doubling element3-109 and dichroic mirror DC₂ may provide an intensity dependent lossmechanism that causes mode locking of the laser. Elements of themode-locked laser that are similar to elements of the mode-locked laserdescribed in connection with FIG. 3-1 are numbered with similarreference numbers and their description is not repeated.

According to some embodiments, a NMM laser cavity may include atrichroic mirror TC₁ that serves as an output coupler and a dichroicmirror DC₂ that serves as a high reflector for a frequency-doubledwavelength λ₂. The trichroic mirror TC₁ may be configured to pass thepump wavelength λ_(p) and be highly reflective for the lasing wavelengthλ₁ and highly reflective for the frequency-doubled wavelength λ₂. Thelaser cavity may include an additional trichroic reflector TC₂ that isconfigured to reflect the pump wavelength back through the gain mediumand pass the lasing wavelength and frequency doubled wavelength. Thelasing wavelength λ₁ may be incident on the frequency-doubling element3-109 where it is converted to the frequency-doubled wavelength λ₂within the laser cavity. The dichroic reflector DC₂ may exhibit a highreflectivity for the frequency-doubled wavelength λ₂. For example, itmay reflect between about 95% and about 100% of the frequency-doubledwavelength, and between about 60% and about 75% of the lasing wavelengthλ₁. Because of the higher loss for the lasing wavelength, the laser willprefer to operate in a mode-locked state having pulses of highintensity, because these high intensity pulses may be converted moreefficiently by the frequency doubling element 3-109 to the doubledfrequency and reflected more efficiently from the dichroic mirror DC₂.The frequency-doubled wavelength λ₂ may then be coupled from themode-locked laser with the dichroic mirror DC₁.

FIG. 3-3C depicts yet another embodiment of a compact, mode-locked laserthat is configured to produce two frequency-doubled output wavelengthsλ₃, λ₄. In some implementations, the gain medium 3-308 may compriseNd:YVO₄ and the coatings on the optical elements in the laser cavity maybe engineered with reflective and transmissive values to providesimultaneous lasing at 1064 nm and 1342 nm wavelengths. Thesewavelengths may be frequency doubled with a doubling element 3-109located external to the laser cavity, for example.

According to some embodiments, a dual-wavelength mode-locked laser maybe arranged similar to the SAM mode-locked laser depicted in FIG. 3-3A.However, the first dichroic mirror is replaced with a trichroic mirrorTC₁, and the second dichroic mirror is replaced with a third trichroicmirror TC₃. Additionally, a gain medium has been selected that may laseat two wavelengths λ₁, λ₂. Further, the saturable absorber mirror 3-325has been modified to exhibit intensity-dependent loss at the two lasingwavelengths.

According to some embodiments, the trichroic mirror TC₁ may beconfigured to efficiently reflect a pump wavelength to the gain medium3-308 and to pass the two lasing wavelengths λ₁, λ₂ to thefrequency-doubling element 3-109. The trichroic mirror TC₃ may beconfigured to reflect the pump wavelength λ_(p) back through the gainmedium 3-308, and to pass the two lasing wavelengths λ₁, λ₂ to the delayelement 3-110 and on to the saturable absorber mirror 3-325. The SAM3-325 and trichroic mirror TC₂ may be end mirrors of the laser cavity.When excited by the pump source, the dual-wavelength laser may mode lockon the two lasing wavelengths.

The mode-locked laser systems depicted in FIG. 3-1 and FIG. 3-3A throughFIG. 3-3C may, or may not, be arranged in a rectilinear configuration asdepicted in the drawings. In some implementations, the cavities may befolded with additional mirrors at various angles and in differentgeometric configurations without departing from the scope of theinvention. The reflective and transmissive coatings formed on theoptical elements will be engineered according to the incidence angles ofthe corresponding lasing, pump, and frequency-doubled beams for whichthe coatings are designed. For example, a coating engineered for highreflectance of a particular wavelength at normal beam incidence willhave a different design for a beam of the same wavelength incident on amirror at 45°. In some embodiments, the coatings may be tailored for aspecific beam incidence angle.

Details of a dual-wavelength saturable absorber mirror 3-325 will now bedescribed. According to some embodiments, a dual wavelength SAM may beformed on a semiconductor substrate 3-405 is depicted in FIG. 3-4A. Asurface of the substrate 3-405 may include a high-reflectance coating3-430. The high-reflectance coating may comprise a multilayer dielectriccoating, in some implementations. In some cases, a high-reflectancecoating may comprise a metallic coating. A first multiple quantum wellstructure 3-412 may be formed on the substrate a distance d₁ from thehigh-reflectance coating. A second multiple quantum well structure 3-410may be formed a second distance d₂ from the high-reflectance coating3-430. According to some embodiments, the first and second multiplequantum well structures may be separated by an intermediatesemiconductor layer 3-407. There may, or may not, be one or moreadditional layers 3-409 formed adjacent the second multiple quantum wellstructure 3-410. Light from the laser cavity may be incident on a firstsurface 3-402 of the saturable absorber mirror.

According to some embodiments, one or more of the substrate 3-405,intermediate layer 3-407 and additional layer or layers 3-409 maycomprise silicon or other semiconductor materials. The multiple quantumwell structures 3-412, 3-410 may be formed by epitaxial growth or atomiclayer deposition, according to some embodiments. The multiple quantumwell structures may be formed from alternating layers of materialshaving compositions comprising one or more of the following elements:In, Ga, As, Al, P.

FIG. 3-4B depicts, according to some embodiments, an energy band-gapdiagram plotted as a function of distance from the high-reflectancesurface 3-430 of the saturable absorber mirror 3-325 depicted in FIG.3-4A. The first multiple quantum well structure 3-412 may create a firstenergy bandgap BG₂, and the second multiple quantum well structure 3-410may create a second energy bandgap BG₄, as depicted in the drawing. Thefirst and second energy bandgaps may be less than the bandgaps BG₁, BG₃,and BG₅ of the surrounding regions. The first energy bandgap BG₂ may beengineered to saturably absorb a first lasing wavelength λ₁, and thesecond energy bandgap BG₄ may be engineered to saturably absorb thesecond lasing wavelength λ₂. The first and second lasing wavelengths maypass through the surrounding regions, having the larger bandgaps, withlittle or no attenuation.

The locations of the first multiple quantum well structure 3-412 andsecond multiple quantum well structure 3-410 may be located toapproximately align with intensity anti-nodes of the first lasingwavelength λ₁ and second wavelength λ₂, respectively, that are reflectedfrom the reflective surface 3-430, as depicted in FIG. 3-4C. Theillustrated intensity anti-nodes 3-442, 3-441 may be located thedistances d₁ and d₂ from the high-reflectance surface 3-430. Theillustrated intensity anti-nodes may not be the only intensityanti-nodes, and there may be more intensity anti-nodes between theillustrated anti-nodes and the high reflectance surface as well asadditional intensity anti-nodes farther from the high reflectancesurface. According to some embodiments, the multiple quantum wellstructure 3-412 having a smaller bandgap BG₂ will be located closer tothe high-reflectance surface 3-430. This may allow the longer wavelengthλ₁ to pass through the second multiple quantum well structure 3-410without being appreciably attenuated. As just one example, the firstmultiple quantum well structure 3-412 may be engineered to have abandgap BG₂ that approximately corresponds to a wavelength of 1342 nm,and the second multiple quantum well structure 3-410 may be engineeredto have a bandgap BG₄ corresponding to approximately 1064 nm. In thismanner, intensity dependent loss can be provided for both lasingwavelengths, so as to produce mode locking at two lasing wavelengths.

Referring again to FIG. 3-3C, when in operation, a dual-wavelength laser3-304 may produce ultrafast pulses at two lasing wavelengths λ₁, λ₂simultaneously. The repetition rate of the pulses at the two differentwavelengths will depend upon the optical path length for each wavelengthwithin the laser cavity. Since there are optical elements in the lasercavity that the lasing beams must pass through (e.g., gain medium 3-308,trichroic mirror TC₃, optical system OS₂, optical delay element 3-110)and since the refractive index in each element may be different for thetwo wavelengths, then the optical path lengths for the first and secondlasing wavelengths within the laser cavity will be different. Adifference in optical path lengths can lead to two different pulserepetition rates, which may be undesirable for some applications.

In some embodiments, the two sets of multiple quantum wells are locatedclose together so that optical radiation from one laser can affectcarrier densities in both quantum wells. The quantum wells may bedesigned to have absorbing states corresponding to λ₁ and λ₂.Cross-saturation of the quantum wells may help synchronize the timing ofpulses from both laser sources.

To avoid producing trains of pulses at two different pulse repetitionrates, the inventors have recognized and appreciated that a compensatingoptical system should be included within the laser cavity to make theoptical path lengths for the two lasing wavelengths approximately equal.Referring to FIG. 3-5A, the inventors have recognized and appreciatedthat a single, path-length-compensating element 3-500, such as an endmirror or output coupler, may be engineered to compensate fordifferences in optical path lengths for two lasing wavelengths within alaser cavity. According to some embodiments, an output coupler mayinclude a first dichroic high-reflectance coating at a first surface3-552 for a first lasing wavelength on a first side of the outputcoupler, and include a second dichroic high-reflectance coating at asecond surface 3-554 on a second side of the output coupler. For anoutput coupler, the reflectivity of each coating for the respectivewavelength may be between about 70% and about 98%, and each coating maytransmit more than 98% of the other wavelength. In embodiments where anend mirror is used as the compensating element, the reflectivity foreach coating may be greater than 98%.

The material and thickness t of the compensating element 3-500 may beselected to compensate for the difference in optical path lengths of thelaser cavity for the first and second lasing wavelengths. As an exampleand without being bound to any particular theory, the thickness of thecompensating element t may be selected according to the followingrelation:

$\begin{matrix}{t = \frac{\delta_{opd}\left( {\lambda_{1},\lambda_{2}} \right)}{n\left( {n_{\lambda\; 1}❘n_{\lambda 2}} \right)}} & (1)\end{matrix}$

where δ_(opt) (λ₁, λ₂) represents the difference in optical path in thelaser cavity for pulses at the first and second lasing wavelengths λ₁,λ₂, and n_(λ1) and n_(λ2) represent the values of group indices for thecompensating element's substrate (between the reflective coatings) forthe first and second lasing wavelengths, respectively. The optical pathdifference δ_(opd)(λ₁, λ₂) may be estimated initially for the lasercavity by measurements to the first surface 3-552 of the output coupler3-500. The pulse-separation interval T may be used to determine a cavitylength more accurately. The first surface may be oriented toward thelaser cavity. Whichever lasing wavelength has the shorter optical pathdifference in the laser cavity will be selected to double-pass throughthe substrate of the compensating element to the second surface 3-554.For example, if pulses at the wavelength λ₂ have a shorter optical pathin the cavity, then the value for n_(λ2) is used in EQ. 1. Pulses thatreflects from the second surface 3-554 pick up additional optical pathin the coupler 3-500, while pulses at the other lasing wavelengthreflect from the first surface 3-552. The additional optical path addedfor pulses at one lasing wavelength may compensate for other opticalpath differences in the laser cavity.

In some cases, the thickness t of the compensating element 3-500 may beless than about 1 mm. Such a thin substrate may not be suitable for ahigh-quality laser-cavity mirror. For example, it may be difficult tomake or retain an optically flat surface (e.g., having a flatness ofλ/10 or better) on a thin substrate. In some embodiments, thecompensating element 3-502 may be formed on, or bonded to, a supportsubstrate 3-556, as depicted in FIG. 3-5B. The support substrate maycomprise an optically flat surface (e.g., having a flatness of λ/10 orbetter) adjacent the compensating element. In some implementations, acompensating element may be optically contacted to, or adhered withoptical adhesive to, the support substrate 3-556.

In some embodiments, a compensating element may be formed on a supportsubstrate 3-556. For example, a first high-reflectance, multilayercoating 3-562 may be formed on the support substrate 3-556. Then, anintermediate layer 3-564 may be deposited to a thickness t. Theintermediate layer may be deposited by a physical deposition process insome embodiments, or by a vapor deposition process in some cases. Insome implementations, the intermediate layer 3-564 may, or may not, bepolished to an optically flat surface after deposition. Subsequently, asecond high-reflectance, multilayer coating 3-566 may be formed on theintermediate layer 3-564.

The first reflective coating of the compensating element toward thelaser cavity may be a dichroic coating that highly reflects a firstlasing wavelength and highly transmits the second lasing wavelength. Forexample, the first reflective coating 3-566 may reflect between about85% and about 98% of the first lasing wavelength λ₁, and may transmitmore than about 98% of the second lasing wavelength λ₂. The secondreflective coating 3-562 may highly reflect the second lasingwavelength, and may, or may not, highly transmit the first lasingwavelength. If a compensating element 3-500, 3-502 is used as a cavityend mirror, the second reflective coating (farthest from the center ofthe laser cavity) may be highly reflective for both lasing wavelengths.Such a coating may be easier and less costly to manufacture. If acompensating element 3-500, 3-502 is used as an output coupler, thesecond reflective coating may be highly reflective for one lasingwavelength and highly transmissive for the other.

The inventors have recognized and appreciated that thermal heatingeffects and/or mechanical stresses on optical elements within the lasercavity can be a significant factor that may influence the performance ofa compact, mode-locked laser. Thermal heating can arise at the pumpsource 3-105, the gain medium 3-107, and/or the frequency-doublingelement 3-109, in some implementations. In regard to the gain medium,the inventors have recognized and appreciated that additional care mustbe taken when mounting the gain crystal. A mount should allow for heatdissipation, and yet avoid mechanically stressing the crystal. Anexample of a mounting structure 3-600 for a gain crystal is shown inFIG. 3-6, according to some embodiments. The depicted mount is designedfor a gain medium having a square cross section, but the mount may bedesigned for other cross-sections such as rectangular or polygonal. Thegain medium may have a length L extending along a direction into thepage.

According to some embodiments, a mounting structure 3-600 for a gainmedium may comprise a first portion 3-620 and a second portion 3-622that are configured to be joined together in a clamping arrangement. Forexample the first portion and second portion may contain through-holes3-640 for screws that allow the two portions to be fastened to andplaced in thermal contact with a supporting base plate. The firstportion 3-620 and the second portion 3-622 may be formed from ahigh-thermal-conducting material such as copper or aluminum, althoughother materials may be used in other embodiments. The first and secondportions may have several interior faces 3-615 that are arranged to beplaced in thermal contact with a gain medium of a laser cavity.According to some embodiments, there may be trenches or openings 3-630located at regions of the mount where corners of the gain medium may belocated (e.g., when the gain medium is mounted in the mounting structure3-600). The trenches or openings 3-630 may reduce mechanical and/orthermal stress that would otherwise be induced on the gain medium. Thetrenches or openings may extend between about 1 mm and about 3 mm oneither side of a corner location of the gain medium. The inventors havefound that the openings at the corners of the gain medium can alleviatethermal and mechanical stress that may otherwise crack the gain mediumand/or adversely affect the optical mode profile of the laser.

In some implementations, the first portion 3-620 and the second portion3-622 of the mounting structure 3-600 may be thermally cooled, e.g.,contacted to thermo-electric coolers. According to some embodiments, thefirst portion may be controllably cooled to a different temperature thanthe second portion or vice versa, so that a temperature gradient may beestablished across the gain medium. Such differential control may beused to steer the laser beam within the laser cavity, e.g., foralignment purposes or for tuning pulsed operation.

The inventors have further recognized and appreciated that mountingstructures that dissipate heat, may adversely affect optical alignmentof a laser cavity. For example, a mounting structure 3-600 for a gainmedium or diode pump source 3-105 may be fastened to a base plate towhich other optical elements of a pulsed laser are fastened. A mountingstructure may dissipate heat into the base plate, and the heat may causeexpansion and/or warping or other distortion of the base plate. As aresult, motion of the base plate can misalign optical elements of thelaser cavity and adversely affect laser performance.

According to some embodiments, a mounting structure or component of apulsed laser that requires significant heat dissipation may be mountedon a partially-isolated platform 3-710, as depicted in plan view in FIG.3-7A. The platform may partially isolate heat dissipation into abaseplate of a pulsed laser. Elevation views of the platform aredepicted in FIG. 3-7B and FIG. 3-7C. A partially-isolated platform 3-710may be formed in a baseplate 3-705 by a machining process, according tosome implementations. For example, the baseplate 3-705 may be part of asolid block of material that is machined to form a housing for acompact, mode-locked laser. One or more trenches or troughs 3-730 may bemachined through the baseplate 3-705 to form the partially-isolatedplatform 3-710. These troughs may extend through the baseplate 3-705, asdepicted in FIG. 3-7C, and partially separate and thermally isolate theplatform 3-710 from the baseplate 3-705. For example, heat cannot bedissipated as readily from the platform into the baseplate.

A plurality of support tabs 3-720 may remain after the machining processthat forms the troughs 3-730. The support tabs provide mechanicalsupport for the platform 3-710, as well as provide partial thermalconduction to the baseplate 3-705. A lower surface of the platform 3-710may be thermally contacted to a thermal-electric cooler (not shown),according to some implementations. In various embodiments, the supporttabs 3-720 are located centrally, with respect to the thickness of theplatform, between upper and lower surfaces of the platform 3-710, asdepicted in FIG. 3-7B. For example, the support tabs 3-720 may belocated in a neutral mechanical plane of the baseplate 3-705 asillustrated in FIG. 3-7B. Locating the support tabs 3-720 centrally withrespect to the thickness of the platform and baseplate can reduce theamount of out-of-plane thermal-mechanical stress imparted between thebaseplate 3-705 and platform 3-710. Reducing the amount of heatdissipated into the baseplate and reducing out-of-plane stress mayreduce warping of the baseplate and undesired relative motion of otheroptical components in the laser cavity. In some embodiments, the supporttabs comprise flexural members that allow the platform to move relativeto the baseplate 3-705, e.g., to accommodate thermo-mechanical stressesinduced by the platform. Motion of some laser components (e.g., the gainmedium 3-107) may not affect operation of the laser as much as othercomponents (e.g., cavity mirrors), and therefore may be tolerated. Thepartial thermo-mechanical isolation of the platform 3-710 can improvethe stability of the laser, and reduce the need for adjustments by askilled operator.

According to some embodiments, one or more platforms 3-710 may be usedto support high temperature elements in a pulsed laser. For example, afirst platform 3-710 may be used to support a diode pump source 3-105 orpump module 2-140, and a second platform may be used to support a lasergain medium 3-107, 1-105. In some implementations, a third platform maybe used to support a nonlinear element 3-109, 2-170.

In some embodiments, multiple pulsed lasers operating at differentcharacteristic wavelengths may be used. The inventors have recognizedand appreciated that pulse trains from two lasers may be synchronizedwithout electro-mechanical feedback control circuitry. In someembodiments, a pulse train from a first mode-locked laser may be used togenerate pulses from a second continuous wave laser, as depicted in FIG.3-8A. A first laser 1-110 a may produce a first train of pulses 3-820 aat a first characteristic wavelength λ₁. Some energy from the pulses maybe converted to the second harmonic via second-harmonic generation (SHG)at a first nonlinear optical element 3-830. Remaining energy at thefundamental wavelength may be directed by a first dichroic mirror DC₁into a second laser 3-800, which comprises a first end mirror DC₂, asecond nonlinear optical element 3-840 for sum-frequency generation(SFG), a gain medium 3-810, and second end mirror DC₃. The end mirrorsmay be dichroic mirrors that are highly reflective for a second lasingwavelength λ₂ and may transmit other wavelengths. For example, the endmirrors may have reflectivity values greater than 99% for the secondlasing wavelength, and may transmit the first lasing wavelength λ₁. Thesecond laser 3-800 may also include a trichroic reflector TC₁ throughwhich a pump wavelength λ_(p) for the gain medium may be introduced intothe cavity.

According to some embodiments, the second laser 3-800 may operate incontinuous-wave mode. Accordingly, the second laser, by itself, willproduce no pulses. Additionally, because the cavity mirrors of thesecond laser have high reflectivity values, the intracavity power can bevery high since the laser does not need to provide power external to thecavity at its operating wavelength λ₂. The high intracavity power maythen be used for sum-frequency generation with pulses injected into thecavity from the first laser 1-110 a to produce a pulse train 3-820 c atthe third wavelength λ₃. Because the second laser 3-800 operates in acontinuous-wave mode, the cavity length of the second laser is not tiedto a pulse repetition rate, so cavity length control may not berequired. Further, since pulse production via SFG is determined bypulses from the first laser 1-110 a, the generated pulses at thesum-frequency wavelength λ₃ are automatically synchronized to the pulsesfrom the first laser, and no electronic synchronization of the two pulsetrains is needed. Synchronization to instrument electronics will stillbe required.

FIG. 3-8B depicts an alternative embodiment of a two-laser system inwhich one laser operates in continuous wave mode. In this system, SFGoccurs before SHG. In some cases, the efficiency of sum-frequencygeneration may be less than second harmonic generation, so that it maybe advantageous to perform SFG first so that the intensity of the firstlaser pulses is higher.

For laser embodiments that employ wavelength conversion via nonlinearoptical elements to obtain a desired wavelength, the nonlinear opticalelements may be supported in mounts that allow angular adjustment of theoptical element with respect to an optical beam axis passing through theoptical element. The angular adjustment may allow the nonlinear elementto rotate to a phase-matching angle for high conversion efficiency.Angular adjustment may be made manually, e.g., by adjustment screws atthe time of manufacture, and then fixed via a glue, resin, or othermethod. In some embodiments, the angular adjustment may not be fixed, sothat a user or technician can make further adjustments when needed.

The inventors have conceived of additional methods for helping tosynchronize pulse trains from two lasers where at least one laserincludes a saturable absorber. FIG. 3-9 depicts a two-laser system 3-900in which a bleaching pulse train 3-820 b from a first mode-locked laser1-110 a is directed to a saturable absorber mirror 3-120 of a secondmode-locked laser 3-910. The second mode-locked laser may comprise again medium 3-810 and output coupler OC₁. The gain medium of the secondlaser may be the same as the gain medium of the first laser.

The bleaching pulse train 3-820 b may be divided from a main outputpulse train 3-820 a of the first laser by a beam splitter BS₁, accordingto some embodiments. As the bleaching pulse train strikes the saturableabsorber mirror, it will assist in bleaching (reducing the optical loss)of the saturable absorber mirror during each pulse. This short reductionin loss will influence the formation and timing of optical pulses 3-820c in the second laser 3-910. In various embodiments, the bleachingpulses should be spatially aligned to the region of the saturableabsorber mirror that is illuminated with the second laser beam. Sincethe optical pulses of the second laser 3-910 will also bleach thesaturable absorber once formed, it is desirable that they strike thesaturable absorber mirror 3-120 simultaneously with pulses from thefirst laser when the two lasers are operating in steady state.Accordingly, an electro-mechanical control circuit 3-920 may be used tocontrol the cavity length (and pulse repetition rate) of the secondlaser.

An example of an electro-mechanical control circuit 3-1000 forcontrolling a cavity length is depicted in FIG. 3-10. Other embodimentsmay use different signal-processing circuitry. In some implementations,pulses from two lasers may be detected with two photodetectors 3-1010,3-1012. The optical pulses may be portions of laser beams tapped offwith beamsplitters, for example, or stray reflections, scatter, orresidual transmission from optical components within the laser cavities.The signals from the photodetectors may be amplified with amplifiers3-1020, 3-1022, and the filtered with low-pass or band-pass filters3-1030, 3-1032. A variable phase delay 3-1034 may be included in onesignal path to allow the two signals to be mixed in quadrature. Theamplifiers may comprise op-amp or radio-frequency amplifiers and may bedigital or analog. The filters may be digital filters or analog filters,and may generate substantially sinusoidal outputs corresponding to thefundamental or harmonic frequencies of the pulse repetition rates forthe two lasers. The outputs from the two filters may then be mixed atmixer 3-1040 to produce sum and difference frequencies.

According to some embodiments, an output from the mixer may be filteredwith a low-pass filter 3-1040 to produce a DC signal, which provides anerror signal proportional to the phase shift between the twofrequencies. The DC signal level may be provided to anelectro-mechanical control circuit 3-920 and monitored to determine howwell cavity lengths are matched. When the cavity lengths are matched,the DC signal level may be near a zero value. When the cavity lengthsare not matched, the magnitude of the DC signal level may increase, andcontrol circuit 3-920 may generate a control signal to an actuator 3-930that moves a cavity end mirror, for example, to decrease the magnitudeof the DC signal level.

In some embodiments, a phase-locked loop may be used instead of a mixer3-1040 in an electro-mechanical control circuit. For example, sinusoidalor digitized square-wave signals from filters 3-1030, 3-1032 may beapplied to a phase detector of a phase-locked loop. An output from thephase detector may be filtered and provided to an electro-mechanicalcontrol circuit 3-920.

II. B. Mode-Locked Semiconductor Lasers

In some implementations, semiconductor laser diodes may be mode lockedto provide a low-cost source of ultrafast pulses. Mode-locked laserdiodes may produce pulses at a desired wavelength (e.g., at blue, green,or red wavelengths) that will be used directly for probing samples ormaking measurements, according to some embodiments. In some cases,pulses produced by a laser diode may be converted to another wavelength(e.g., frequency doubled) for use in probing or measuring applications.For example, a mode-locked laser diode may produce pulses at infraredwavelengths, and these pulses may be frequency doubled to the blue,green, or red regions of the optical spectrum.

One embodiment of a mode-locked laser diode 4-100 is depicted in FIG.4-1. A mode-locked semiconductor laser may comprise a laser diode 4-105and a saturable absorber mirror 3-120. The ends of the laser cavity maybe defined by a reflective coating 4-112 formed on one end of thesemiconductor laser diode 4-105 and the saturable absorber mirror 3-120,according to some embodiments. The laser cavity may include a firstoptical system OS₁ that reshapes and/or changes the divergence of anoptical beam from the laser diode. The laser cavity may further includea second optical system OS₂ that may reshape and/or focus theintra-cavity beam onto the saturable absorber mirror. In someembodiments, the laser cavity may include an optical delay element3-110. The optical delay element may be any embodiment of a delayelement described above in connection with FIG. 3-2A through FIG. 3-2D.A mode-locked laser diode may lase at a wavelength λ₁ and produce atrain of ultrafast pulses with durations shorter than about 100 ps.

In some implementations, a laser diode 4-105 may include opticalcoatings on either end of an optical waveguide structure. The opticalcoatings 4-110, 4-112 may be formed by any suitable deposition process,such as a vapor deposition process or a physical deposition process. Insome implementations, a first end of the laser diode may include apartially-transmissive coating 4-112 that serves as an output couplerfor the laser cavity. The transmissive coating 4-112 may transmit aportion of the lasing beam outside the cavity to provide a train ofultrafast pulses. The transmittance of the coating 4-112 may be betweenapproximately 2% and approximately 15%, according to some embodiments,and its reflectivity may be between about 98% and about 85%. An oppositeend of the laser diode 4-105 may be coated with an anti-reflectioncoating 4-110, so as to allow most of the radiation from the laser diodeto pass into the laser cavity without significant reflection. Forexample, the anti-reflection coating 4-110 may reflect less than 1% ofthe lasing wavelength λ₁.

In some embodiments, the saturable absorber for a mode-locked laserdiode 4-200 may be integrated with a semiconductor laser diode on a samechip, as depicted in FIG. 4-2. For example, a saturable absorber 4-665may be integrated onto a substrate on which the laser diode 4-620 isformed. The laser cavity may comprise an optical system OS₁ thatreshapes and/or changes the divergence of the beam from the laser diode.In some embodiments, the optical system OS₁ may be the only opticalsystem in the laser cavity that is used to change the shape and/ordivergence of the beam in the cavity. The laser cavity may also includean optical delay element 3-110 and an output coupler OC₁. The outputcoupler may comprise a beam splitter that transmits a portion of thelasing beam outside the cavity and reflects most of the lasing beam backwithin the laser cavity. The transmittance of the output coupler OC₁ maybe between approximately 2% and approximately 15%, according to someembodiments. As described above, one end of the laser diode 4-620 thatis opposite the saturable absorber 4-665 may include an anti-reflectioncoating. The saturable absorber may include a high reflective coatingthat reflects the majority of radiation from the laser diode back intothe laser cavity.

Another embodiment of a mode-locked laser diode 4-300 is depicted inFIG. 4-3. In this embodiment, an optical fiber 4-320 is used as anoptical delay element for the laser cavity. The laser cavity may includea saturable absorber 4-665 and a high-reflectance coating adjacent thesaturable absorber that are integrated onto a same substrate as a laserdiode 4-620, according to some embodiments. The laser cavity may furtherinclude an optical coupling component 4-310 that is used to coupleradiation from the laser diode 4-620 into the optical fiber 4-320. Anoptical output coupling element 4-330 may be located at a second end ofthe fiber 4-320, and be configured as an output coupler for the lasercavity, according to some embodiments.

In some implementations, the optical coupling element 4-310 may compriseoptical adhesive. For example the optical fiber 4-320 may be aligned andadhered to an end of the laser diode using the optical adhesive. Thefiber end may be bonded at a location where radiation from a waveguidingregion of the laser diode couples more efficiently into the fiber. Insome embodiments, the optical coupling element 4-310 may comprise a balllens or a graded refractive index (GRIN) lens. According to someembodiments, a surface of the output optical coupling element 4-330, atan opposite end of the optical fiber, may include a reflective coating4-332, so as to provide output coupling from the laser cavity. Theoutput coupling element 4-330 may comprise a ball lens or GRIN lens insome implementations. In some embodiments, the output coupling element4-330 may comprise a lens mounted near an end of the optical fiber4-320.

Any of the depicted embodiments of mode-locked laser diodes illustratedin FIG. 4-1 through FIG. 4-3 may, or may not, include a wavelengthconversion element 3-109. According to some embodiments, a wavelengthconversion element may comprise a frequency-doubling crystal that isaligned to a beam from the laser cavity, or may include a nonlinearelement employed for parametric conversion or four-wave mixing. In someembodiments, a nonlinear element may comprise a periodically-poledmaterial, such as lithium niobate, that may be integrated onto a samesubstrate as the laser diode.

The use of mode-locked laser diodes may be advantageous for someembodiments that do not require high amounts of power, for example,power levels exceeding about 300 mW. One advantage of mode-locked laserdiodes is their compact size and a reduction in the number of opticalelements used in the laser. Because the lasing medium can be very small(e.g., less than 5 mm in width), it may be possible to use arrays ofmode-locked laser diodes in some embodiments. In some implementations,an array of mode-locked laser diodes may share common optical elements.For example, two or more laser diodes may share one or more opticalelements (e.g., one or more of an optical delay element 3-110, opticalsystems OS₁, OS₂, and saturable absorber mirror 3-120).

II. C. Mode-Locked Fiber Lasers

According to some embodiments, ultrafast pulses may also be producedusing mode-locked fiber lasers. Some examples of mode-locked fiberlasers are depicted in FIG. 5-1 through FIG. 5-3. A mode-locked fiberlaser may include optical elements that are used in diode-pumpedsolid-state lasers, as described above and depicted in FIG. 3-3A throughFIG. 3-3C. However, in a mode-locked fiber laser the gain mediumcomprises a length of optical fiber 5-120 that can also provide anoptical delay element for the laser cavity. According to someembodiments, a diode pump source 3-105 may provide a pumping wavelengthλ_(p) that is coupled into an end of the fiber 5-120, as depicted inFIG. 5-1. A fiber-laser cavity may be defined by a first dichroic endmirror DC₁ and a saturable absorber mirror 3-120 that causes passivemode locking of the fiber laser, in some implementations.

Referring to FIG. 5-1 and according to some embodiments, a mode-lockedfiber laser 5-100 may comprise a first optical system OS₁ that isconfigured to couple an output beam from a diode pump source 3-105 intoan optical fiber 5-120 that serves as a gain medium for the laser. Insome implementations, the beam from the diode pump source 3-105 may becoupled into the cladding of the optical fiber to excite the core andgain medium of the optical fiber 5-120. A second optical system OS₂ maybe arranged to couple radiation from the optical fiber, e.g., to form abeam at a lasing wavelength λ₁. The laser cavity may further include adichroic mirror DC₂ positioned near or at an end of the optical fiber5-120, as depicted in the drawing. The second dichroic mirror DC₂ maytransmit a majority of the lasing wavelength λ₁ to the saturableabsorber mirror 3-120, and reflect most of the pump wavelength λ_(p)back through the optical fiber. For example, the second dichroic mirrorDC₂ may transmit more than about 98% of the lasing wavelength andreflect more than about 98% of the pump wavelength. A third dichroicmirror DC₃ may be included outside the laser cavity between the pumpsource and the optical fiber, and may be used to direct an output laserbeam from the fiber laser 5-100. The third dichroic mirror may transmita majority (e.g., more than about 98%) of the pump wavelength λ_(p) andreflect a majority (e.g., more than about 98%) of the lasing wavelengthλ₁, according to some implementations.

Another embodiment of a mode-locked fiber laser 5-200 is depicted inFIG. 5-2. In some implementations, optical coupling elements may befabricated or bonded at opposing ends of the optical fiber 5-120. Forexample, a first optical element 5-210 may be bonded to or formed on afirst end of the optical fiber. The first optical element may comprise aball lens or a graded refractive index lens that is attached directly,or attached with a supporting structure, to an end of the optical fiber.Additionally, the first optical element 5-210 may include a dichroiccoating that transmits a majority (e.g., more than about 98%) of thepump wavelength λ_(p) and reflects a majority (between about 98% andabout 85%) of the lasing wavelength λ₁. Accordingly, the first opticalelement 5-210 may comprise an output coupler for the fiber laser 5-200.

The second optical element 5-220 may comprise a dichroic coating formedon an end of the optical fiber, in some embodiments, that is engineeredto transmit a majority (e.g., more than about 98%) of the lasingwavelength λ₁ and reflect a majority (e.g., more than about 98%) of thepump wavelength λ_(p) back into the optical fiber. In some embodiments,the second optical element 5-220 may comprise a ball lens or a GRIN lensthat is attached directly, or coupled with a supporting structure, to anend of the optical fiber. For example, a GRIN lens may be adhered to anend of the fiber with an optical adhesive, and an exposed end of theGRIN lens may be coated with a dichroic coating that is engineered totransmit a majority (e.g., more than about 98%) of the lasing wavelengthλ₁ and reflect a majority (e.g., more than about 98%) of the pumpwavelength λ_(p) back into the optical fiber. According to someembodiments, there may be a first optical lens system OS₁ that is usedto couple pump radiation from the laser diode 3-105 into the opticalfiber, and a second optical lens system OS₂ that is used to focusradiation from the optical fiber onto the saturable absorber mirror3-120.

FIG. 5-3 depicts yet another embodiment of a mode-locked fiber laser5-300. Such an embodiment may comprise fewer optical elements than inthe previous embodiments of fiber lasers described above. According tosome implementations, the fiber laser cavity may be defined by anoptical prism 5-310 located at one end of the optical fiber 5-120 and asaturable absorber mirror 3-120 located at an opposite end of theoptical fiber. The optical prism 5-310 may include a first surface thatis covered with a first dichroic coating 5-312. The first dichroiccoating may transmit a majority (e.g., more than about 98%) of the pumpsource wavelength λ_(p) and reflect a majority (e.g., more than about98%) of the lasing wavelength λ₁. A second surface of the optical prism5-310 may include a second dichroic coating 5-314 that is configured totransmit majority (e.g., more than about 98%) of the pump wavelengthλ_(p) and reflect the majority (e.g., between about 85% and about 98%)of the lasing wavelength λ₁ back into the optical fiber. The seconddichroic coating 5-314 may serve as an output coupler for the fiberlaser. For example the second dichroic coating 5-314 may transmitbetween approximately 2% and approximately 15% of the lasing wavelengthλ₁. According to some embodiments, there may be an output couplingelement 5-220 located at an opposing end of the optical fiber 5-120. Theoutput coupling element 5-220 may couple lasing radiation from the fiberto the saturable absorber mirror 3 120. In some embodiments, the fiberoutput coupling element may comprise a ball lens or a graded refractiveindex lens that is adhered to an end of the optical fiber. In someimplementations, the output optical coupling element 5-220 may include adichroic coating that is engineered to transmit a majority of the lasingradiation λ₁ and reflect a majority of the pump radiation λ_(p) backinto the fiber. The output optical coupling element 5-220 may couplelasing radiation λ₁ to and from the saturable absorber mirror 3-120, andmay or may not be in contact with the SAM.

II. D. Gain-Switched Lasers

In some embodiments, gain-switched lasers may be employed as a pulsedlaser 1-110 for an analytical instrument 1-100. Gain-switched laserstypically having longer pulses than mode-locked lasers, but can haveless complexity and be manufactured at lower cost. Gain-switched lasersmay be useful when fluorescent lifetimes for the samples have longerdecay rates (e.g., greater than about 5 ns).

The inventors have conceived of pulser circuits and techniques forproducing short and ultrashort optical pulses from laser diodes andlight-emitting diodes. The pulsing circuits and techniques have beenemployed, in some implementations, to gain-switch semiconductor lasersand produce a train of −85 picosecond (ps) pulses (FWHM) having peakpowers of approximately 1 W at repetition rates of up to 100 MHz (T asshort as 10 nanoseconds). In some embodiments, a unipoloar or bipolarcurrent waveform may be produced by a pulser circuit and used to drive alaser diode's gain medium in a manner to excite optical pulses andsuppress emission at the tails of the pulses. In some embodiments, aunipoloar or bipolar current waveform may be produced by a pulsercircuit and may be used to drive one or more light-emitting diodes tooutput short or ultrashort optical pulses.

For purposes of describing gain switching in laser diodes, FIGS. 6-1Athrough 6-1C are included to illustrate laser dynamics associated withgain switching. FIG. 6-1A illustrates a pump-power curve 6-110 that isrepresentative of pump power applied to a gain medium of a gain-switchedlaser, according to some embodiments. As depicted, the pump power may beapplied for a brief duration (depicted as approximately 0.6microseconds) to the gain medium in a laser cavity. For a semiconductorlaser diode, application of pump power may comprise applying a biascurrent across a p-n junction or multiple quantum wells (MQWs) of thelaser diode. The pump power pulse may be applied repetitively atregularly-spaced time intervals, for example, at a pulse-separationinterval or pulse repetition time T.

During application of the pump power pulse, optical gain in the lasercavity increases until the gain begins to exceed optical losses in thecavity. After this point, the laser may begin to lase (i.e., amplifyphotons passing through the gain medium by the process of stimulatedemission). The amplification process results in a rapid increase inlaser light and depletion of excited states in the gain medium toproduce at least one output pulse 6-130 as depicted. In someembodiments, the pump power pulse 6-110 is timed to turn off atapproximately the same time that the peak of the output pulse occurs.Turning off the pump power pulse terminates further lasing, so that theoutput pulse 6-130 quenches. In some embodiments, the output pulse 6-130may have a shorter duration than the pump pulse 6-110, as depicted inthe drawing. For example, an output pulse 6-130 produced by gainswitching may be less than ⅕ the duration of the pump pulse 6-110.

If the pump power pulse is not turned off, then the dynamics depicted inFIG. 6-1B may occur. In this case, the pump power curve (shown as pumpcurrent density) 6-140, depicted as a step function, represents currentdensity applied to a semiconductor laser. The graph shows that the gainmedium is excited by a pumping current density, which produces a carrierdensity N in the gain region of the laser diode. The pump currentdensity I of about twice a lasing threshold current density I_(th), isapplied at time t=0, and is then left on. The graph shows the increasein carrier density N for the semiconductor gain region until the opticalgain of the laser exceeds loss in the cavity. After this point, a firstpulse 6-161 builds up, depleting the carrier density and optical gain toa value less than the cavity loss, and is emitted. Subsequently, asecond pulse 6-162 builds up, depletes carrier density N, and isemitted. The build-up and depletion of carrier density repeats forseveral cycles until the laser stabilizes into continuous wave operation(e.g., after about 7 nanoseconds in this example). The cycle of pulses(pulse 6-161, pulse 6-162, and subsequent pulses) are referred to asrelaxation oscillations of the laser.

The inventors have recognized and appreciated that a challenge whengain-switching a laser to produce ultrashort-pulses is to avoiddeleterious effects of continued relaxation oscillations. For example,if a pump power pulse 6-110 is not terminated quickly enough, at least asecond optical pulse 6-162 (due to relaxation oscillation) may begin tobuild up in the laser cavity and add a tail 6-172 to a gain-switchedoutput pulse 6-170, as depicted in FIG. 6-1C. The inventors haverecognized and appreciated that such a tail can be undesirable in someapplications, such as applications aimed at distinguishing fluorescentmolecules based on fluorescent lifetimes. If the tail of an excitationpulse is not reduced sufficiently quickly, excitation radiation mayoverwhelm a detector unless wavelength filtering is employed.Alternatively or additionally, a tail on an excitation pulse maycontinue to excite a fluorescent molecule and may complicate detectionof fluorescent lifetime.

If the tail of an excitation pulse is reduced sufficiently quickly,there may be negligible excitation radiation present during fluorescentemission. In such implementations, filtering of the excitation radiationduring detection of fluorescent emission may not be needed to detect thefluorescent emission and distinguish fluorescent molecule lifetimes. Insome cases, the elimination of excitation filtering can significantlysimplify and reduce the cost of an analytic system 1-160 as well asallow a more compact configuration for the system. For example, when afilter is not needed to suppress the excitation wavelength duringfluorescent emission, the excitation source and fluorescent detector canbe located in close proximity (e.g., on a same circuit board orintegrated device, and even within microns of each other).

The inventors have also recognized and appreciated that in some cases, atail on an excitation pulse may be tolerated. For example, an analyticsystem 1-160 may have an optical configuration that easily allows forincorporation of a wavelength filter into a detection optical path. Thewavelength filter may be selected to reject excitation wavelengths, sothat a detector receives quantifiable fluorescence from a biologicalsample. As a result, excitation radiation from the pulsed optical sourcedoes not overwhelm the detected fluorescence.

In some embodiments, a fluorescent molecule's emission lifetime τ may becharacterized by a 1/e intensity value, according to some embodiments,though other metrics may be used in some embodiments (e.g., 1/e²,emission half-life, etc.). The accuracy of determining a fluorescentmolecule's lifetime is improved when an excitation pulse, used to excitethe fluorescent molecule, has a duration that is less than thefluorescent molecule's lifetime. Preferably, the excitation pulse has aFWHM duration that is less than the fluorescent molecule's emissionlifetime by at least a factor of three. An excitation pulse that has alonger duration or a tail 6-172 with appreciable energy may continue toexcite the fluorescent molecule during a time when decaying emission isbeing evaluated, and complicate the analysis of fluorescent moleculelifetime. To improve fluorescent lifetime determination in such cases,deconvolution techniques may be used to deconvolve the excitation pulseprofile from the detected fluorescence.

In some cases, it may be preferable to use ultrashort-pulses to excitefluorescent molecules in order to reduce quenching of the fluorescentmolecule or sample. It has been found that extended pumping of afluorescent molecule may bleach and/or damage the fluorescent moleculeover time, whereas higher intensities for shorter durations (even thoughfor a same total amount of energy on the molecule) may not be asdamaging to the fluorescent molecule as the prolonged exposure at lowerintensity. Reducing exposure time may avoid or reduce photo-induceddamage to fluorescent molecules, and increase the amount of time ornumber of measurements for which the fluorescent molecules may be usedin an analytic system 1-160.

In some applications, the inventors have found it desirable for theexcitation pulse to terminate quickly (e.g., within about 250 ps fromthe peak of the pulse) to a power level that is at least about 40 dBbelow the peak power level of the pulse. Some embodiments may toleratesmaller amounts of power reduction, e.g., between about 20 dB and about40 dB reduction within about 250 ps. Some embodiments may requiresimilar or higher amounts of power reduction within about 250 ps, e.g.,between about 40 dB and about 80 dB in some embodiments, or betweenabout 80 dB and about 120 dB in some embodiments. In some embodiments,these levels of power reduction may be required within about 100 ps fromthe peak of the pumping pulse.

According to some embodiments, the pulse-separation interval T (see FIG.1-2) may also be an important aspect of a pulsed laser system. Forexample, when using a pulsed laser to evaluate and/or distinguishemission lifetimes of fluorescent molecules, the time between excitationpulses is preferably longer than any emission lifetime of the examinedfluorescent species in order to allow for sufficiently accuratedetermination of an emission lifetime. For example, a subsequent pulseshould not arrive before an excited fluorescent molecule or ensemble offluorescent molecules excited from a previous pulse has (or have) had areasonable amount of time to fluoresce. In some embodiments, theinterval T needs to be long enough to determine a time between anexcitation pulse that excites a fluorescent molecule and a subsequentphoton emitted by the fluorescent molecule after termination ofexcitation pulse and before the next excitation pulse.

Although the interval between excitation pulses T should be long enoughto determine decay properties of the fluorescent species, it is alsodesirable that the pulse-separation interval T is short enough to allowmany measurements to be made in a short period of time. By way ofexample and not limitation, emission lifetimes (1/e values) offluorescent molecules used in some applications may be in the range ofabout 100 picoseconds to about 10 nanoseconds. Therefore, depending onthe fluorescent molecules used, a pulse-separation interval as short asabout 200 ps may be used, whereas for longer lifetime fluorescentmolecules a pulse-separation interval T greater than about 20nanoseconds may be used. Accordingly, excitation pulses used to excitefluorescence for fluorescent lifetime analysis may have FWHM durationsbetween about 25 picoseconds and about 2 nanoseconds, according to someembodiments.

In some applications, such as fluorescent lifetime imaging, where anintegrated time-domain imaging array is used to detect fluorescence andprovide data for lifetime analysis and a visual display, thepulse-separation interval T may not need to be shorter than a frame rateof the imaging system. For example, if there is adequate fluorescentsignal following a single excitation pulse, signal accumulation overmultiple excitation pulses for an imaging frame may not be needed. Insome embodiments, a pulse repetition rate R_(p) of the pulsed opticalsource 1-110 may be synchronized to a frame rate R_(f) of the imagingsystem, so that a pulse repetition rate may be as slow as about 30 Hz.In other embodiments, the pulse repetition rate may be appreciablyhigher than the frame rate, and fluorescent decay signals for each pixelin an image may be integrated values following multiple excitationpulses.

An example of a gain-switched pulsed laser 6-200 is depicted in FIG.6-2A. According to some embodiments, a pulsed laser 6-200 may comprise acommercial or custom semiconductor laser diode 6-201 formed on asubstrate 6-208. A laser diode may be packaged in a housing 6-212 thatincludes an electrical connector 6-224. There may be one or more opticalelements 6-205 (e.g., one or more lenses) included with the package toreshape and/or change the divergence of an output beam from the laser.The laser diode 6-201 may be driven by a pulser circuit 6-210 which mayprovide a sequence of current pulses over a connecting cable 6-226 andat least one wire 6-220 to the diode 6-201. The drive current from thepulser circuit 6-210 may produce a train of optical pulses 6-222 emittedfrom the laser diode.

According to some embodiments, a laser diode 6-201 may comprise asemiconductor junction comprising a first layer 6-202 having a firstconductivity type (e.g., p-type) and a second layer 6-206 having anopposite conductivity type. There may be one or more intermediate layers6-204 formed between the first and second layers. For example, theintermediate layers may comprise multiple-quantum-well (MQW) layers inwhich carriers injected from the first and second layers recombine toproduce photons. In some embodiments, the intermediate layers mayinclude electron and/or hole blocking layers. The laser diode maycomprise inorganic materials and/or organic semiconductor materials insome implementations. The materials may be selected to obtain a desiredemission wavelength. For example and for inorganic semiconductors,III-nitride compositions may be used for lasers emitting at wavelengthsless than about 500 nm, and III-arsenide or III-phosphide compositionsmay be used for lasers emitting at wavelengths greater than about 500nm. Any suitable type of laser diode 6-201 may be used including, butnot limited to, a vertical cavity surface emitting laser (VCSEL), anedge-emitting laser diode, or a slab-coupled optical waveguide laser(SCOWL).

According to some embodiments, one or more pulsed LEDs may be usedinstead of a gain-switched laser diode. Pulsed LEDs may be useful fortime-of-flight, 3-D imaging, and fluorescent imaging applications. AnLED may have a lower intensity than a LD, so multiple LEDs may be used.Because an LED does not undergo relaxation oscillations or dynamicsassociated with lasing action, its output pulses may be of longerduration and have a wider spectral bandwidth than would occur for alaser. For example, the output pulses may be between about 100 ps andabout 2 ns, and the spectral bandwidth may be about 20 nm or larger. Insome implementations, output pulses from an LED may be between about 100ps and about 500 ps. Longer excitation pulses may be acceptable forexcitation of fluorescent molecules that have longer decay times.Additionally, an LED may produce an unpolarized or partially polarizedoutput beam. The embodiments of pulser circuits described below may beused to drive one or more LEDs in some implementations of pulsed opticalsources.

One advantage of using LEDs is their lower cost compared to laserdiodes. Additionally, LEDs provide a broader, typically incoherent,spectral output that can be better suited for imaging applications(e.g., an LED may produce less optical interference artifacts). For alaser diode, the coherent radiation can introduce speckle in imagingapplications, unless measures are taken to avoid speckle in thecollected images. Also, LEDs can extend excitation wavelengths into theultraviolet (e.g., down to about 240 nm), and can be used for excitingautofluorescence in biological samples.

The inventors have recognized that some conventional laser diode systemscomprise current driver circuitry that can be modeled as depicted inFIG. 6-2B. For example, the current driver 6-210 may comprise a pulsedvoltage source 6-230 configured to deliver current pulses to a laserdiode. Connection to the laser diode is typically made through a cable6-226, adaptor or connector 6-224, and a single wire 6-220 that isbonded to a contact pad on the laser diode 6-210. The connection betweenthe adaptor 6-224 and laser diode may include a series inductance L1 andseries resistance R1. The connection may also include small junctioncapacitances (not shown) associated with contacts and/or the diodejunction.

The inventors have recognized and appreciated that increasing the numberof wire bonds (e.g., between the connector 6-224 and laser diode 6-201)may reduce the inductance and/or resistance of the connection to a laserdiode 6-201. Such a reduction in inductance and/or resistance may enablehigher speed current modulation of the laser diode and shorter outputpulses. According to some embodiments, a single wire bond 6-220 may bereplaced with multiple parallel wire bonds to improve the speed of alaser diode. For example, the number of wire bonds may be increased tothree or more. In some implementations, there may be up to 50 wire bondsto a laser diode.

The inventors have investigated the effects of increasing the number ofwire bonds 6-220 on a commercial laser diode. An example commerciallaser considered was an Oclaro laser diode, model HL63133DG, nowavailable from Ushio, of Cypress, Calif. Results from numericalsimulations of increasing a number of wire bonds are illustrated in FIG.6-2C. The simulation increased the number of wire bonds from a singlebond for the commercial device (curve 6-250) to three wire bonds (curve6-252) and to 36 wire bonds (curve 6-254). The average drive currentdelivered to the laser diode for a fixed 18V pulse was determined over arange of frequencies for the three different cases. The results indicatethat a higher number of wire bonds allows more current to be deliveredto the laser diode at higher frequencies. For example, at 1 GHz, the useof just three wire bonds (curve 6-252) allows more than four times asmuch current to be delivered to the laser diode than for a single wirebond. Since short and ultrashort pulses require higher bandwidth (higherfrequency components to form the short pulse), adding multiple wirebonds allows the higher frequency components to drive the laser diode ina shorter pulse than a single wire bond. In some implementations, themultiple wire bonds may extend between a single contact pad or multiplecontact pads on a laser diode and an adaptor or connector 6-224 on alaser diode package. The connector may be configured for connection toan external, standardized cable (e.g., to a 50-ohm BNC or SMA cable).

In some embodiments, the number of wire bonds and the wire bondconfiguration may be selected to match an impedance of the adaptorand/or cable connected to the laser diode. For example, the impedance ofthe wire bonds may be matched to the impedance of a connector 6-224 toreduce power reflections from the laser diode to the current driver,according to some embodiments. In other embodiments, the impedance ofthe wire bonds may intentionally mismatch the diode's input impedance.The mismatch may generate a negative pulse between positivecurrent-driving pulses. Selecting a packaging method for a laser diode(e.g., selecting a number of wire bonds to a laser diode from anadaptor) may improve the current modulation supplied to the laser diodeat higher frequencies. This can make the laser diode more responsive tohigh-speed gain-switching signals, and may enable shorter opticalpulses, faster reduction of optical power after the pulse peak, and/orincreased pulse repetition rates.

Referring now to FIG. 6-3, the inventors have further recognized andappreciated that applying a bipolar pulse waveform 6-300 to a laserdiode may suppress an undesired emission tail 6-172 (see FIG. 6-1C) onproduced optical pulses. A bipolar pulse may also be used to shorten anoptical pulse from an LED. A bipolar pulse may comprise a first pulse6-310 of a first polarity followed by a second pulse 6-312 of anopposite polarity. The magnitude of the second pulse 6-312 may bedifferent from the magnitude of the first pulse. In some embodiments,the second pulse may have a magnitude that is approximately equal to orless than the first pulse 6-310. In other embodiments, the second pulse6-312 may have a magnitude that is greater than the first pulse 6-310.

In some embodiments, the magnitude of the second pulse may be betweenabout 10% of the magnitude of the first pulse and about 90% of themagnitude of the first pulse. In some implementations, the magnitude ofthe second pulse may be between about 25% of the magnitude of the firstpulse and about 90% of the magnitude of the first pulse. In some cases,the magnitude of the second pulse may be between about 50% of themagnitude of the first pulse and about 90% of the magnitude of the firstpulse. In some embodiments, an amount of energy in the second pulse maybe between about 25% of an amount of energy in the first pulse and about90% of the energy in the first pulse. In some implementations, an amountof energy in the second pulse may be between about 50% of an amount ofenergy in the first pulse and about 90% of the energy in the firstpulse.

The first drive pulse may forward bias a laser diode junction andthereby generate carriers in the diodes active region that may recombineto produce an optical pulse. The second drive pulse 6-312, opposite inpolarity, may reverse bias the diode junction and accelerate removal ofcarriers from the active region to terminate photon generation. When thesecond electrical pulse 6-312 is timed to occur at approximately thesame time as, or just before (e.g., within about 200 ps), the secondrelaxation oscillation pulse (see pulse 6-162 of FIG. 6-1B), the carrierconcentration that would otherwise produce the second optical pulse isdiminished so that the emission tail 6-172 is suppressed.

Various circuit configurations may be used to produce bipolar pulsewaveforms. FIG. 6-4A depicts just one example of a circuit that may beused to drive a laser diode or one or more LEDs with a bipolar pulsewaveform. In some embodiments, a transmission line 6-410 (e.g., a stripline or co-axial conductor assembly) may be configured in a pulsercircuit 6-400 to deliver bipolar pulses to a semiconductor laser diode6-420 or at least one LED. The transmission line 6-410 may be formed ina U-shaped configuration and biased on a first conductor by a DC voltagesource V_(DD) through a charging resistor R_(ch). The transmission linemay have an impedance that approximately matches the impedance of alaser diode, according to some embodiments. In some embodiments, thetransmission line's impedance may be approximately 50 ohms. In someimplementations, the transmission line's impedance may be betweenapproximately 20 ohms and approximately 100 ohms. In someimplementations, the transmission line's impedance may be betweenapproximately 1 ohm and approximately 20 ohms.

The pulser 6-400 may further include a terminating resistor Z_(term)connected between the second conductor of the transmission line at oneend of the transmission line and a reference potential (e.g., ground inthe depicted example). The other end of the second conductor of thetransmission line may be connected to the laser diode 6-420. The ends ofthe transmission line's first conductor may connect to a switch M₁(e.g., a field effect transistor or bipolar junction transistor) thatcan be activated to periodically shunt the ends of the first conductorto a reference potential (e.g., ground).

In some instances, the terminating impedance Z_(term) may beapproximately equal to the impedance of the transmission line 6-410 inorder to reduce reflections back into the line. Alternatively, theterminating impedance Z_(term) may be less than the impedance of theline in order to reflect a negative pulse into the line (after shuntingby switch M₁) and to the laser diode 6-420. In some implementations, theterminating impedance Z_(term) may include a capacitive and/or inductivecomponent selected to control the shape of the reflected negative pulse.A transmission line pulser, as depicted in FIG. 6-4A, may be used toproduce electrical bipolar pulses having a repetition rate within arange between about 30 Hz to about 200 MHz. According to someembodiments, a transmission line 6-410 for a transmission line pulsermay be formed on a printed circuit board (PCB), as depicted in FIG.6-5A.

FIG. 6-4B depicts an embodiment of a driver circuit 6-401 connected toan optical semiconductor diode 6-423 (e.g., a laser diode or one or moreLEDs) that may be formed using discrete components, and that may beintegrated onto a substrate (such as a chip or PCB). In someembodiments, the circuit may be integrated onto a same substrate as alaser diode or LED 6-423. The laser driver circuit 6-401 may comprise acontrol input 6-405 connected to the gate or base of a transistor M1.The transistor may be a CMOS FET, a bipolar junction transistor, or ahigh-electron mobility transistor (such as a GaN pHEMT), though otherhigh-speed, high current handling transistors may be used. Thetransistor may be connected between a current source 6-430 and areference potential (e.g., a ground potential, though other referencepotential values may be used). The transistor M₁ may be connected inparallel between the current source 6-430 and reference potential withthe laser diode 6-423 (or one or more LEDs) and a resistor R₁ that isconnected in series with the laser diode. According to some embodiments,the driver circuit 6-401 may further include a capacitor C₁ connected inparallel with the resistor R₁ between the laser diode and referencepotential. Though a transistor M₁ is described, any suitablecontrollable switch having a high conductive and low conductive statemay be used.

In operation, the driver circuit 6-401 may provide a current thatbypasses the laser diode 6-423 when the transistor M₁ is on, or in aconducting state. Therefore, there is no optical output from the laserdiode. When the transistor M₁ switches off, current may flow through thelaser diode due to the increased resistive path at the transistor. Thecurrent turns the laser diode on, until the transistor is switched onagain. Light pulses may be generated by modulating the control gate ofthe transistor between on and off states to provide current pulses tothe laser diode. This approach can reduce the amount of voltage on thesupply and the voltage on the transistor needed to drive the lasercompared to some pulsing techniques, which is an important aspect forimplementation of such high-speed circuits.

Due to the presence of the resistor R₁ and parallel capacitor C₁, chargewill build up on the capacitor when the diode is forward conducting.This can occur when the transistor M₁ is in an “off” state, e.g., a low-or non-conducting state. When the transistor is turned on, the voltagestored across the capacitor will reverse bias the laser diode. Thereverse bias effectively produces a negative pulse across the laserdiode, which may reduce or eliminate the emission tail 6-172 that wouldotherwise occur without the negative pulse. The value of the resistor R₁may be selected such that substantially all of the charge on thecapacitor will discharge before the switch is subsequently opened and/ora subsequent light pulse is generated by the laser diode. For example,the time constant t₁=R₁C₁ may be engineered to be less than aboutone-half or one-third of the pulse repetition interval T. In someimplementations, the time constant t₁=R₁C₁ may be between approximately0.2 ns and approximately 10 ns.

In some implementations, the transistor M₁ may be configured to switchto a conducting state after a first peak of an output light pulse fromthe laser diode. For example, and referring to FIG. 6-1B, an opticaldetection and logic circuit may sense the decaying intensity of thefirst pulse 6-161 and trigger the transistor M₁ to switch to aconducting state. In some embodiments, the transistor M₁ may betriggered to switch to a conducting state based on a stable clock signal(e.g., triggered with reference to a synchronizing clock edge). In someimplementations, the transistor M₁ may be triggered to switch to aconducting state according to a predetermined delay time measured fromthe time at which the transistor M₁ switches to a non-conducting state.Switching the transistor M₁ to a conducting state at a selected time mayreduce the laser power shortly after the peak light pulse, shorten thelaser pulse, and/or reduce tail emission of the pulse.

Although the drive circuit shown in FIG. 6-4B shows the current source6-430 located on the anode side of the laser, in some embodiments acurrent source may be located alternatively, or additionally, on thecathode side of the laser (e.g., connected between the transistor M1,resistor R₁, and a reference potential such as ground).

Other embodiments of drive circuitry for producing ultrashort-pulses arepossible. For example, a current pulse drive circuit 6-402 for a laserdiode or LED may comprise a plurality of current drive branchesconnected to a node of a laser diode, as depicted in FIG. 6-4C. Thedriver circuit 6-402 may be formed using discrete or integratedcomponents and integrated onto a substrate (e.g., an ASIC chip or PCB).In some embodiments, the driver circuit may be integrated onto a samesubstrate as one or more optical semiconductor diodes 6-425 (e.g., alaser diode or one or more light-emitting diodes). Although the drawingdepicts the driver circuit as connected to the anode of the laser diode6-425, in some embodiments similar drive circuitry may alternatively, oradditionally, be connected to the cathode of the laser diode. Drivecircuitry connected to the cathode side of the laser diode may employtransistors of an opposite type and voltage sources of opposite polaritythan those used on the anode side of the laser diode.

According to some implementations, there may be N circuit branches(e.g., circuit branches 6-432, 6-434, 6-436) configured to apply Nforward-bias current pulses to a laser diode 6-425 or LED and M circuitbranches (e.g., circuit branch 6-438) configured to apply M reverse-biascurrent pulses to the laser diode. In FIG. 6-4C, N=3 and M=1, thoughother values may be used. Each forward-bias current branch may comprisea voltage source V_(i) configured to deliver a forward-bias current tothe laser diode. Each reverse-bias current branch may comprise a voltagesource V₃ configured to deliver a reverse-bias current to the laserdiode. Each circuit branch may further include a resistor R_(i)connected in series with a switch or transistor Mi. Each circuit branchmay include a capacitor C_(i) connected on one side to a node betweenthe transistor Mi and resistor R_(i), and connected on the other side toa fixed reference potential. In some embodiments, the capacitance C_(i)may be junction capacitance associated with the transistor Mi (e.g,source-to-body capacitance), and a separate discrete capacitor may notbe provided. In some implementations, at least one additional resistormay be included in series with the diode 6-425 to limit the amount oftotal current delivered from the circuit branches.

In operation, timed and pulsed control signals may be applied to thecontrol inputs S_(i) of the switches or transistors M₁, so as togenerate a sequence of current pulses from each of the circuit branchesthat are summed and applied across the laser diode junction. The valuesof components in each branch (V_(i), V_(j), R_(i), C_(i)) and the timingand pulse duration of control pulses applied to the control inputs S_(i)can be independently selected to produce a desired bipolar current pulsewaveform that is applied to the laser diode 6-425. As just one example,the values of V₁, V₂, and V₃ may be selected to have different values.The values of R₁, R₂, and R₃ may be the same, and the values of C₁, C₂,and C₃ may be the same. In this example, the staggering of pulsedsignals to the control inputs S_(i) may produce a staggered sequence ofoverlapping current pulses from the forward-bias circuit branches thathave similar pulse durations but different pulse amplitudes. A timedpulse from the reverse-bias circuit branch may produce a current pulseof opposite polarity that can quench or rapidly turn off theforward-biasing pulse, and may further produce a reverse-biasing pulsethat can suppress tail emission from the laser diode. Thereverse-biasing pulse may be timed carefully, so that it at leastpartially overlaps temporally with one or more of the forward-biasingpulses. Accordingly, the circuit depicted in FIG. 6-4C may be used tosynthesize bipolar current pulses as depicted in FIG. 6-3.

FIG. 6-4D depicts another embodiment of a pulse driver 6-403, which maybe manufactured using radio-frequency (RF) components. The RF componentsmay be designed to handle signals at frequencies between about 50 MHzand about 1 GHz, according to some embodiments. In some implementations,a pulse driver 6-403 may comprise an input DC block 6-435, which ACcouples an input waveform (e.g., a square wave or sinusoidal wave) tothe driver. The DC block may be followed by an amplifier 6-440, whichproduces non-inverted and inverted output waveforms that proceed alongseparate circuit paths 6-440 a, 6-440 b, respectively. The first circuitpath 6-440 a may include one or more adaptors 6-442. A variable phaseshifter 6-445 may be included in the second circuit path 6-440 b toselectively phase shift the signal in the second path with respect tothe signal in the first path.

The first and second circuit paths may connect to non-inverting inputsof an RF logic gate 6-450 (e.g., an AND gate or other logic gate).Inverting inputs of the logic gate 6-450 may be terminated with suitableimpedance-matched terminators 6-446 to avoid spurious power reflectionsat the gate. The non-inverting and inverting outputs of the logic gate6-450 may connect to a combiner 6-460 along two circuit paths 6-450 a,6-450 b. The inverted circuit path 6-450 b may include a delay element6-454 and attenuator 6-456, either or both of which may be adjustable.The delay element may be used to delay the inverted signal with respectto the non-inverted signal, and the attenuator may be used to adjust theamplitude of the inverted signal.

The resulting inverted signal and non-inverted signal from the logicgate may then be summed at the combiner 6-460. The output from thecombiner 6-460 may be connected to an RF amplifier 6-470 that providesoutput bipolar pulses to drive a laser diode or one or more LEDs. Theoutput bipolar pulses may have a waveform as depicted in FIG. 6-4E.

In operation, an input square wave or sinusoidal wave may be AC coupledinto the driver and split into the two circuit paths 6-440 a, 6-440 b asnon-inverted and inverted versions. The first amplifier 6-440 may be alimiting amplifier that squares up a sinusoidal waveform, according tosome embodiments. In the second circuit path 6-440 b the invertedwaveform may be phase shifted with an adjustable phase shifter 6-445 totemporally delay the inverted waveform with respect to the non-invertedwaveform. The resulting waveforms from the first amplifier 6-440 maythen be processed by the RF logic gate 6-450 (e.g., an AND gate) toproduce short RF pulses at the non-inverting and inverting outputs ofthe logic gate. The duration of the short RF pulses may be adjustedusing the phase shifter 6-445, according to some embodiments. Forexample, the phase shifter may adjust a time period during which boththe non-inverted waveform and inverted waveform at the input to a logicAND gate 6-450 are simultaneously in an “on” state, which will determinethe length of the output pulses.

Referring to FIG. 6-4E, the short inverted pulses 6-417 from the logicgate 6-450 may be delayed an amount 8 by the delay element 6-454 withrespect to the non-inverted pulses 6-415 and attenuated by attenuator6-456 to a desired amplitude before being combined with the non-invertedpulse. In some embodiments, the negative-pulse magnitude |V_(p−)| may beless than the positive-pulse amplitude V_(p+). The pulse-separationinterval T may be determined by the frequency of the sinusoidal orsquare wave input into the pulse driver 6-403. The output pulse waveformmay, or may not, include a DC offset. Although the output waveform isdepicted as having a square-shaped waveform, capacitances andinductances in the RF components and/or cabling may produce outputpulses having more rounded waveforms, more like the waveform depicted inFIG. 6-3.

As mentioned earlier in connection with FIG. 6-4C and FIG. 6-4B, theapplication of current or voltage to a laser diode or LED can be to boththe anode and cathode of a diode in some embodiments. A radio-frequencypulse driver circuit 6-404 that can apply a split or differentialvoltage or current pulse to both the cathode and anode of a diode isdepicted in FIG. 6-4F. The front end of the circuit may be similar tothe front end of the pulse driver circuit 6-403 depicted in FIG. 6-4D,according to some embodiments. However, in the pulse driver circuit6-404 the non-inverted and inverted outputs from the logic gate 6-450may not be combined and instead applied as a differential drive to theanode and cathode of the laser diode. For simplification, the circuitryassociated with producing a subsequent negative or reverse biasing pulseis not shown in FIG. 6-4F.

An example of a split or differential drive produced by the differentialpulse driver circuit 6-404 is depicted in FIG. 6-4G. A first output fromthe logic gate 6-450 may produce a positive pulse 6-416 of amplitude+V_(p), and a second inverted output from the logic gate 6-450 mayproduce a negative pulse 6-418 of opposite amplitude −V_(p). The pulsetrains may, or may not, have a small DC offset in some embodiments. Thepresence of the positive pulse 6-416 and negative pulse 6-418 produce aforward biasing pulse across the laser diode having an effectiveamplitude 2V_(p). By splitting the bias across the laser diode andapplying a partial bias to the anode and to the cathode, the amplitudeof voltage pulses handled by the pulse driver 6-404 may be effectivelyreduced by a factor of 2. Accordingly, the pulse driver 6-404 mayoperate at a higher frequency and produce shorter pulses than it mightotherwise be able to achieve for higher amplitude pulses. Alternatively,a pulse driver circuit 6-404 may effectively double the amplitude of thedriving pulse applied across a laser diode compared to a driving circuitthat only provides a biasing pulse +V_(p) to the anode of the laserdiode. In such embodiments, the power output from the laser diode may beincreased.

Another way in which power applied to the laser diode and/or drivingspeed may be increased is depicted in FIG. 6-4H. According to someembodiments, a plurality of pulse-driver outputs 6-470 may be connectedto an anode of a laser diode 6-425 or LED. In this example, four pulsedrivers are connected to the anode of the laser diode. In someembodiments, in which differential pulse driver circuitry is used, theremay be multiple drivers connected to the cathode of the laser diode aswell. Each driver and its associated cabling may have an impedance Z₀,and a laser diode 6-425 may have been impedance Z_(L). Because of theirparallel connection, the output impedances of the drivers are divided bythe number of drivers connected to the laser diode. The power deliveredinto the diode may be increased when the combined impedances of thepulse drivers is approximately matched to the impedance of the laserdiode 6-425, or vice versa.

The graph in FIG. 6-41 illustrates the increase in efficiency of powercoupled into the laser diode 6-425 for four driving sources as afunction of the impedance of the laser diode and the laser diodecircuit. In the example, the four pulse drivers each have a lineimpedance of about 50 ohms and are configured to deliver an output pulseof 5 V amplitude with a maximum current of approximately 100 mA. Theplot shows that the power coupled into the laser diode reaches a maximumwhen the laser diode's impedance is at approximately 10 ohms. This valueis approximately equal to the parallel output impedance of the fourpulse driver outputs 6-470. Accordingly, the impedance of the laserdiode 6-425 and its associated circuitry may be designed toapproximately match the combined impedance of one or more pulse driversused to drive the laser diode, according to some embodiments.

Other circuit driver configurations may be used to pulse laser diodes orlight-emitting diodes. According to some embodiments, a currentinjection into a light-emitting diode may be pulsed to producesub-nanosecond pulses using a pulser circuit described in “A simplesub-nanosecond ultraviolet light pulse generator with high repetitionrate and peak power,” authored by P. H. Binh et al., Rev. Sci. Instr.Vol. 84, 083102 (2013), or in “An ultraviolet nanosecond light pulsegenerator using a light emitting diode for test of photodetectors”authored by T. Araki et al., Rev. Sci. Instr. Vol. 68, 1365 (1997).

Another example of a pulser circuit is depicted in FIG. 6-4J. Accordingto some embodiments, a pulser circuit may comprise a pulse generator6-480, which may receive one or more clock signals from a system clock,for example, and output a train of electrical pulses to a driver circuit6-490 that injects current pulses into a laser diode or light-emittingdiode responsive to the received electrical pulses from the pulsegenerator. Accordingly, the output optical pulses may be synchronized tothe system clock. The system clock may also be used to operate detectionelectronics (e.g., an imaging array).

According to some embodiments, the pulse generator 6-480 may be formedfrom a combination of passive and digital electronic components, and maybe formed on a first circuit board. In some cases, a pulse generator mayinclude analog circuit components. In other embodiments, a portion ofthe pulse generator may be formed on a same board as the driver circuit6-490, and a portion of the pulse generator may be formed on a separateboard remote from the driver circuit. The driver circuit 6-490 may beformed from passive, analog, and digital electronic components, and maybe formed on a same or different circuit board as the pulse generator orportion of the pulse generator. An optical source (laser diode orlight-emitting diode) may be included on a circuit board with the drivercircuit, or may be located in a system and connected to the drivercircuit 6-490 by high-speed cabling (e.g., SMA cables). In someimplementations, the pulse generator 6-480 and driver circuit 6-490 mayinclude emitter-coupled logic elements. According to some embodiments,the pulse generator 6-480, driver circuit 6-490, and opticalsemiconductor diode 6-423 may be integrated onto a same printed circuitboard, laminate, or integrated circuit.

An example of a pulse generator 6-480 is depicted in FIG. 6-4K. In someimplementations, a pulse generator may include a first stage thatproduces two differential clock outputs, one delayed with respect to theother. The first stage may receive a clock input and include a fan-out6-481 and delay 6-483. The fan-out may comprise logic drivers and logicinverters arranged to produce two copies of the clock signal and twoinverted copies of the clock signal. According to some embodiments, theclock may have a symmetric duty cycle, though asymmetric duty cycles maybe used in other embodiments. One copy and one inverted copy may form adifferential clock output (CK1, CK1 ) and may be delayed by a delayelement 6-483 with respect to a second copy and second inverted copy(CK2, CK2 ). The delay element may comprise any suitable variable orfixed delay element. Examples of delay elements include RF delay linesand logic gate delays. In some implementations, the first pair of clocksignals (CK1, CK1 ) is delayed at least a fraction of a clock cycle withrespect to the second pair of clock signals (CK2, CK2 ). A delay mayinclude one or more full cycles in addition to a fractional cycle.Within each pair of clock signals, the inverted signal may besynchronized to its counterpart so that rising and falling edges of theclocks occur at essentially the same time.

The inventors have found that ultrashort pulsing of a laser diode or LEDcan be controlled more reliably by adjusting a length of acurrent-driving pulse from the pulse generator 6-480 and maintaining afixed amplitude rather than adjusting an amplitude of an ultrashortcurrent-driving pulse. Adjusting the length of the current-driving pulseadjusts an amount of energy delivered to the laser diode per pulse. Insome embodiments, high-speed circuits allow for high-resolution controlof signal phase (e.g., by adjusting a delay or phase with an analog ordigital delay element 6-483), which can be used to obtainhigh-resolution control of pulse length, according to someimplementations.

In some cases, the first stage of the pulse generator 6-480 may comprisea dual-output clock instead of the fan-out 6-481 and delay 6-483. Adual-output clock may generate two differential clock signals, andprovide adjustable phase delay between the two differential clocksignals. In some implementations, the adjustable phase delay may have acorresponding time resolution as little as 3 ps.

Regardless of how the delayed clock signals CK1, CK2 and their inversesare produced, the signals may be transmitted over high-speedtransmission lines to a high-speed logic gate 6-485. For signaltransmission over cables between boards, the clock pulses maydeteriorate due to cabling. For example, limited bandwidth oftransmission lines may distort the clock pulses differently and resultin unequal timing. In some implementations, a same type of cabling ortransmission line may be used for all the clock signals, so thattransmission distortions affect the four clock signals equally. Forexample, when signal distortions and timing offsets are essentially thesame for the four clock signals, a resulting driving pulse produced bythe receiving logic gate 6-485 will be essentially the same as it wouldbe if there were no signal distortions from transmission of the clocksignals. Accordingly, transmission of clock signals over distances ofseveral feet may be tolerated without affecting the driving-pulseduration. This can be useful for producing ultrashort driving pulsesthat are synchronized to a system clock and have finely adjustable pulseduration (e.g., adjustable in increments of about 3 ps). If the clocksignals are produced locally (e.g., on a same board as the drivercircuit 6-490), signal distortions associated with transmission of theclock signals may not be significant and the transmission lines maydiffer to some extent.

According to some embodiments, the clock signals may be AC coupled withcapacitors C₁ and provided to data inputs of a high-speed logic gate6-485. Capacitors C₁ may have a capacitance between about 10 nF andabout 1 μF. According to some embodiments, the logic gate may comprisean emitter-coupled logic (ECL), two-input, differential AND/NAND gate.An example of logic gate 6-485 includes model MC100EPO5 available fromON Semiconductor of East Greenwich, R.I. The AC-coupled signals at thedata inputs to the logic gate may appear similar to the signals depictedin FIG. 6-4L, where the horizontal dashed line indicates a zero voltagelevel. The depictions in FIG. 6-4L do not include distortions introducedby transmission lines. The distortions may round and alter the shapes ofthe signal profiles, but may not affect the relative phases of the clocksignals when a same type and length of cabling is used for each clocksignal. Delay element 6-483 may provide a delay Δt indicated by thevertical dashed lines, which may be adjustable in increments as small as3 ps. In some implementations, a delay element 6-483 may provide anadjustable delay in increments having a value between 1 ps and 10 ps.Logic gate 6-485 may process the received clock signals and produce anoutput signal at an output port Q corresponding to the delay introducedby delay element 6-483. With a small delay, the output comprises asequence of short or ultrashort pulses. With a high-speed logic gate6-485, the pulse durations may be between about 50 ps and about 2 ns(FWHM) in some embodiments, between about 50 ps and about 0.5 ns in someembodiments, between about 50 ps and about 200 ps in some embodiments,and yet between about 50 ps and about 100 ps in some embodiments. Thedriving pulses from port Q may have a substantially square profile dueto high-speed slew rates of the ECL logic gate 6-485. A biasing circuit6-487 may be connected to the output port Q, and a voltage V₁ appliedfor positive emitter-coupled logic. Output pulses provided from anoutput terminal P_(out) of the pulse generator 6-480 may include a DCoffset, according to some embodiments.

In some implementations, two or more high-speed logic gates 6-485 may beconnected in parallel between capacitors C₁ and the bias circuit 6-487.The logic gates may be the same, and operate in parallel to providegreater current driving capability at an output of the pulse generator.The inventors have recognized and appreciated that the logic gate 6-485,or gates, need to provide high speed switching (i.e., fast rise and falltimes to produce ultrashort driving pulses), and need to provide enoughoutput current to drive a high current transistor M₁ in the drivercircuit 6-490. In some implementations, connecting logic gates 6-485 inparallel provides improved performance of the pulser circuit, and allowsproduction of sub-100-ps optical pulses.

FIG. 6-4M depicts an embodiment of a driver circuit 6-490, which may beconnected to a laser diode or LED 6-423. A driver circuit may include anAC-coupled input, having a capacitor C₂ in series with a resistor R₃,connected to a gate of a high-speed transistor M1. Capacitance of C₂ maybe between approximately 0.1 μF and approximately 10 μF, according tosome embodiments, and R₃ may have a value between approximately 10 ohmsand approximately 100 ohms. Transistor M₁ may comprise ahigh-electron-mobility field-effect transistor (HEMT FET) capable ofswitching high currents (e.g., at least one ampere and, in some cases,up to four amps or more), according to some embodiments. Transistor M₁may be a high-speed transistor capable of switching such large currentsat multi-gigahertz speeds. According to some embodiments, transistor M₁may switch more than 1 amp for an electrical pulse duration betweenabout 50 ps and about 2 ns at a repetition rate between 30 Hz andapproximately 200 MHz. An example of transistor M₁ includes modelATF-50189-BLK available from Avago Technologies of San Jose, Calif.Biasing and filtering circuit elements (e.g., resistors R₄, R₇, and C₃)may be connected between capacitor C₂ and the gate of transistor M1. Thedrain of transistor M₁ may be directly connected to a cathode of a laserdiode or light-emitting diode 6-423, and a source of transistor M₁ mayconnect to a reference potential (e.g., ground). The anode of diode6-423 may connect to a diode voltage source V_(LD). A resistor R6 andcapacitor C₄ may be connected in parallel across diode 6-423. Accordingto some embodiments, resistor R6 may have a value between approximately50 ohms and approximately 200 ohms, and C₄ may have a capacitancebetween approximately 5 pF and approximately 50 pF. A capacitor C₅(having a value between approximately 1 μF and approximately 5 μF) mayalso be connected between the diode voltage source V_(LD) and areference potential (e.g., ground) in parallel with the diode 6-423 andtransistor M1.

In some embodiments, a protection diode (not shown) may be connected ina reverse direction across the cathode and anode of the laser diode6-423. The protection diode may protect the laser diode from excessivereverse bias potential that could break down the laser diode junction.

In operation, a pulse from the pulse generator 6-480 momentarily turnson transistor M1, allowing current to be injected into the active regionof laser diode or light-emitting diode 6-423. In some implementations, alarge amount of forward current (e.g., up to four amps) flows throughtransistor M1 briefly. The forward current injects carriers into thelaser diode junction and produces a short or ultrashort pulse of opticalradiation. When transistor M1 turns off, parasitic inductances continuethe flow of current across the light-emitting diode or laser diode,building up charge on the cathode side of the diode, until it can bedissipated by the RC network connected in parallel with the laser diode.This temporary build-up of charge at the cathode provides a reverse biaspulse to the laser diode, and accelerates removal of carriers from theactive region. This accelerates termination of the optical pulse.

The inventors have found that the optical pulsing technique describedfor the embodiment of FIG. 6-4M is superior to pulsing techniques basedon differentiating square-wave pulses, because it can provide a higherand shorter current pulse that may be required to turn on a laser diode.

The inventors have assembled various pulse driving circuits and haveused them to drive laser diodes. FIG. 6-5A depicts another embodiment ofan assembled pulser circuit 6-500. This embodiment implements a pulser6-400 as depicted in FIG. 6-4A. In the assembled circuit, thetransmission line 6-410 is formed as a parallel-plate strip linepatterned in a U-shaped configuration on a printed circuit board, asdepicted in the figure. A GaN pHEMT transistor was used as a shuntingswitch M1 to short two ends of the U-shaped transmission line. Thepulser circuit 6-500 can be operated at repetition rates of up to 100MHz and used to drive a 50 ohm load. In some embodiments, a pulsercircuit may be operated at repetition rates between approximately 10 MHzand approximately 1 GHz.

A measured waveform from the pulser 6-500 is depicted in FIG. 6-5B. Thewaveform shows a positive pulse having an amplitude of approximately19.5 V followed by a negative pulse that reaches an amplitude ofapproximately −5 V following the positive pulse. The duration of thepositive pulse is approximately 1.5 nanoseconds. Referring again to FIG.6-4A, the pulser 6-500 was constructed to a have a terminating resistorZ_(term) of approximately 50 ohms and a pull-up or charging resistorR_(c)h of approximately 200 ohms. The value of Z_(term) was chosen toreduce power reflections from the terminating resistance back into thetransmission line. The bias applied to the transmission line 6-410 was100 V, and the switch M₁ was driven at a repetition rate of 100 MHz.Approximately −1.3 V of DC bias was coupled to the diode via a bias tee,to tune the relative offset from 0 V bias. The driving pulse for theswitch M₁ was a square-wave signal oscillating between approximately 0 Vand approximately 2 V.

A commercial test-bed driver was used to drive a commercial laser diode(Ushio model HL63133DG) to produce sub-100-ps optical pulses. Opticalpulse measurements are shown in FIG. 6-5C and FIG. 6-5D. As shown inFIG. 6-5C, pulses with reduced tail emission were produced at arepetition rate of 100 MHz. The average power from the laser diode wasmeasured to be about 8.3 milliwatts. The pulse duration, shown in FIG.6-5D, was measured to be approximately 84 picoseconds. The intensity ofthe optical emission from the laser diode was found to be reduced byapproximately 24.3 dB approximately 250 ps after the peak of the pulse.Even though the laser diode had a single bond wire to the diode,sub-100-ps pulses were produced. Shorter pulses (e.g., between about 25ps and about 75 ps) may be produced with multiple bond wires or withfurther improvements to the pulser circuit.

FIG. 6-6A depicts one example of a semiconductor laser 6-600 that may beused to produce optical pulses by gain switching, according to any ofthe above-described gain-switching apparatus and techniques. The laserand pulse driving circuitry may be mass produced and manufactured atlow-cost. For example, the laser may be microfabricated as anedge-emitting device using planar integrated circuit technology. Such alaser may be referred to as a slab-coupled optical waveguide laser(SCOWL). The drawing depicts an end-on, elevation view of the laser. Thelaser may be formed from a GaAs/AlGaAs material system (e.g., to emitradiation in the green, red, or infrared regions of the opticalspectrum), but other material systems (such as GaN/AlGaN) may be used insome implementations (e.g., to emit radiation in the green, blue, orultraviolet regions of the spectrum). Laser diodes may be manufacturedfrom other semiconductor material systems that include, but are notlimited to: InP, AlInGaP, InGaP, and InGaN.

According to some embodiments, a SCOWL may be formed on an n-typesubstrate or buffer layer 6-627 (e.g., a GaAs substrate or GaAs layerthat may comprise Al). For example, a buffer layer may compriseAl_(x)Ga_(1-x)As where x is between approximately 0.25 and approximately0.30. The refractive index of the substrate or base layer may have afirst value n₁ that is between about 3.4 and 3.5, according to someembodiments. An electron-transport layer 6-617 of low-doped n-typesemiconductor material may be formed on the substrate 6-627. In someembodiments, the electron-transport layer 6-617 may be formed byepitaxial growth to comprise Al_(x)Ga_(1-x)As where x is betweenapproximately 0.20 and approximately 0.25 and have an n-type dopantconcentration of approximately 5×10¹⁶ cm³. The thickness h of theelectron-transport layer may be between about 1 micron and about 2microns. The transport layer 6-617 may have a second value of refractiveindex n₂ that is greater than n₁. A multiple quantum well region 6-620may then be formed on the electron-transport layer 6-617. The multiplequantum well region may comprise alternating layers of materials (e.g.,alternating layers of AlGaAs/GaAs) having different dopingconcentrations that modulate energy bandgaps in the MQW region. Thelayers in the quantum well region 6-620 (which may have thicknessesbetween approximately 20 nm and approximately 200 nm) may be depositedby epitaxy, atomic layer deposition, or a suitable vapor depositionprocess. The multiple quantum well region may have an effective thirdvalue of refractive index n₃ that is greater than n₂. A hole-transportlayer 6-615 of p-type doped material may be formed adjacent the quantumwell region, and have a value of refractive index n₄ that is less thann₂. In some embodiments, the values of refractive index for thedifferent regions of a SCOWL may be as illustrated in FIG. 6-6B,according to some embodiments. In some embodiments, a SCOWL may compriseGaN semiconductor and its alloys or InP semiconductor and its alloys.

After the layers of the laser device have been deposited, trenches 6-607may be etched into the layers to form an active region of the laserhaving a width w that is between about 0.25 micron and about 1.5microns. An n-contact 6-630 may be formed on a first surface of thedevice, and a p-contact 6-610 may be formed on the p-type transportlayer 6-615, adjacent the active region. Exposed surfaces of thesemiconductor layers may be passivated with an oxide or otherelectrically insulating layer, according to some embodiments.

The trenches 6-607 adjacent the active region, and the values ofrefractive indices n₁, n₂, n₃, and n₄ confine the optical mode of thelaser to a lasing region 6-625 that is adjacent to the quantum wells andunder the devices central rib, as depicted in the drawing. A SCOWL maybe designed to couple higher-order transverse modes, that mightotherwise form and lase in the lasing region 6-625, to lossyhigher-order slab modes in adjacent regions. When designed properly, allhigher-order transverse modes from the lasing region 6-625 have highrelative loss compared to the fundamental mode in the lasing region andwill not lase. In some implementations, the transverse optical mode ofthe SCOWL 6-600 may be a single transverse mode. The width of theoptical mode may be between approximately 0.5 micron and approximately 6microns. A mode profile 6-622, taken in the x direction, may be shapedas depicted in FIG. 6-6B, according to some embodiments. In otherimplementations, a SCOWL may produce multiple optical transverse modesthat are provided to an analytical instrument 1-100. The length of theactive region (along a dimension into the page) may be between 20microns and 10 mm, in some embodiments. The output power of the SCOWLmay be increased by selecting a longer length of the active region. Insome embodiments, a SCOWL may deliver an average output power of morethan 300 mW.

Although a semiconductor laser (e.g., a SCOWL) and pulser circuitry maybe combined to make a low-cost, ultrafast, pulsed laser suitable formany applications, the turn-off rate shown in FIG. 6-5D may not besuitable for some fluorescent lifetime analyses. In some cases, a morerapid turn-off may be needed. For example, the inventors have found thatsome measurements based on fluorescent lifetime may require the tail ofthe pulse to extinguish to a level between approximately 25 dB andapproximately 40 dB below the pulse peak within 250 ps after the pulsepeak. In some cases, the pulse power may need to drop to this range ofvalues within 100 ps after the pulse peak. In some implementations, thepulse tail may need to drop to a level between approximately 40 dB andapproximately 80 dB below the pulse peak within 250 ps after the pulsepeak. In some implementations, the pulse tail may need to drop to alevel between approximately 80 dB and approximately 120 dB below thepulse peak within 250 ps after the pulse peak.

One approach for further suppressing the emission tail of a pulse is toinclude a saturable absorber with a pulsed laser or high-brightness LEDsystem. According to some embodiments, a semiconductor saturableabsorber 6-665 may be incorporated onto a same substrate as asemiconductor laser 6-600 or high-brightness LED, as depicted in FIG.6-6C. The semiconductor laser may comprise a SCOWL structure thatincludes a quantum well region 6-620, according to some embodiments. TheSCOWL may be driven with a pulsed source 6-670, such as a pulser circuit6-400 or other pulsing circuit described above.

Adjacent to one end of the SCOWL, a saturable absorber 6-665 may beformed. The saturable absorber 6-665 may comprise a region having aband-gap that is tailored to absorb photons from the semiconductorlaser. For example, the saturable absorber may comprise a single quantumwell or multiple quantum wells that have at least one energy band gapthat is approximately equal to a characteristic energy of the laser'soptical emission. In some embodiments, a saturable absorber may beformed by ion implanting a region of the laser diode, so as toelectrically isolate the region within the laser diode cavity. Anegative bias may be applied to the region to encourage absorptionrather than gain for the same laser diode structure. At high fluencefrom the laser 6-600, the valence band of the saturable absorber maybecome depleted of carriers and the conduction band may fill, impedingfurther absorption by the saturable absorber. As a result, the saturableabsorber bleaches, and the amount of radiation absorbed from the laseris reduced. In this manner, the peak of a laser pulse may “punchthrough” the saturable absorber with a smaller attenuation in intensitythan the tail or wings of the pulse. The tail of the pulse may then besuppressed further with respect to the peak of the pulse.

According to some embodiments, a high reflector (not shown) may beformed or located at one end of the device. For example, the highreflector may be located at one end of the laser, farthest from thesaturable absorber, so as to redirect laser emission through thesaturable absorber and increase output power. According to someembodiments, an anti-reflection coating may be applied to an end of thesaturable absorber and/or SCOWL to increase extraction from the device.

According to some embodiments, a saturable absorber may include abiasing supply 6-660. The biasing supply may be used to sweep carriersout of the active region after each pulse and improve the response ofthe saturable absorber. In some embodiments, the bias may be modulated(e.g., at the pulse repetition rate) to make the saturable recovery timebe time-dependent. This modulation may further improve pulsecharacteristics. For example, a saturable absorber can suppress a pulsetail by differentially higher absorption at low intensity, if therecovery time of the saturable absorber is sufficient. Such differentialabsorption can also reduce the pulse length. The recovery time of asaturable absorber may be adjusted by applying or increasing a reversebias to the saturable absorber.

II. E. Direct Modulation of Laser Output

The inventors have recognized and appreciated that it is also possibleto make ultrafast pulses from a continuous-wave laser by directmodulation of the laser's output. Direct modulation of the laser'soutput may be done, in some embodiments, using a switching array 7-100of cascaded optical switches, as depicted in FIG. 7-1A. According tosome embodiments, the optical switches 7-105 may be connected by opticalfibers or optical waveguides 7-102, and control signals may be appliedto control inputs 7-103 of the optical switches. In someimplementations, the switching array 7-100 may be integrated onto asingle substrate, e.g., as an integrated array of waveguides andelectro-optic switches such as lithium niobate switches.

The optical switches 7-105 in the switching array may be configured toreceive an optical signal at an input port 7-101 and switch the opticalsignal between a first output port P1 and a second output port P2 at afirst switch S1. In some embodiments, the switching of the opticalsignal may be implemented by applying a drive signal at a control input7-103 of the optical switch S1. For example, the drive signal may applyan electric field to an electro-optical element of the switch. In someembodiments, an optical switch 7-105 may include two input ports,although only one input port 7-101 is depicted in the drawing.

In some implementations, an optical switch 7-105 may comprise aMach-Zehnder interferometric switch that may be controlledelectro-optically, responding to a control input signal applied to aninput port 7-103. For example, one optical path of the Mach-Zehnderinterferometer may include a length of lithium niobate to which anelectric field is applied responsive to the control signal. The appliedelectric field may change the refractive index of the lithium niobateand thereby change the optical path length in that arm of theinterferometer. Accordingly, application of an applied electric fieldmay change an output signal from a first port P1 to a second port P2,and thereby be used to switch the input optical energy back-and-forthbetween the two output ports rapidly.

According to some embodiments, a control signal applied to a controlinput 7-103 may be a square wave, for example, though in someembodiments sinusoidal control signals may be used. The application ofthe square wave to an optical switch may effectively modulate the outputpower that flows from one of its output ports (e.g., as light isdirected into and away from the port). Stated alternatively andreferring to FIG. 7-1B, the insertion loss of the switch, as viewedthrough an output port, modulates between a low value (e.g., an on state7-131) and a high value (e.g., an off state 7-132) responsive to theapplied control signal. Such modulations in loss as viewed from anoutput port are depicted in FIG. 7-1B for optical switches S1, S2, S4,S8, S9 along an upper branch of the array 7-100. In this example,switches S4, S8, and S9 are depicted as being controlled together andstaggered in time from the modulations of switches S1 and S2.

In some embodiments, an optical switch in an on state 7-131 may exhibitan insertion loss between about 0 dB and about 3 dB. In someimplementations, an optical switch in an off state 7-132 may increasethe insertion loss by about 20 dB or more. According to someembodiments, an optical switch in an off state 7-132 may exhibit a lossbetween about 15 dB and about 25 dB.

The modulations of insertion losses for the switches lead tocorresponding modulations in output intensities from ports of theswitching array 7-100, as depicted in FIG. 7-1C. For example, theapplication of a square wave to a first switch S1 may modulate theintensity output from its first port P1 between a low value and ahigh-value, as depicted in the top trace of FIG. 7-1C. In operation, theintensity received at the input port 7-101 of the first switch S1alternates as output pulses 7-135 between the two output ports P1 and P2due to the switching action. According to some implementations, thetiming of the control signals to successive switches along a cascadedpath may be different than the timing for a preceding switch. Forexample, the timing of the control signal for the second switch S2 maybe delayed in time with respect to the control signal for the firstswitch S1, as indicated in FIG. 7-1B. The second switch S2 may operatein the same manner as the first switch, however its switching action maybe offset in time with respect to the first switch S1. As a result, thesecond switch S2 will alternate the power received at its input (fromthe output port P1 of switch S1) between its output ports P3 and P4.

The timing of loss modulation (as viewed through port P3) for the secondswitch S2 is depicted in the middle trace of FIG. 7-1B, and depicts thetiming offset from the modulations of the first switch S1. Thecorresponding intensity of light that is received from the output portP3 of the second switch is depicted in the middle trace of FIG. 7-1C. Ina similar manner, the timing of a control signal applied to the thirdswitch S4 in the optical path is offset in time as depicted in FIG. 7-1Bat the lower trace. Accordingly, the optical pulse received from anoutput port P8 of the switching array 7-100 is further shortened asdepicted in FIG. 7-1C at the lower trace. As indicated by the drawings,the cascading of the two switches with offset control signals andmodulations reduces the pulse length of a received input pulse byapproximately one-half for each successive switch in an optical path forswitches operating with even duty cycles.

In the diagrams of FIG. 7-1B and FIG. 7-1C, the on-to-off ratio orextinction ratio of the switches has been artificially reduced to show abackground noise level 7-140. In practice, the extinction ratio of theoptical switches may be appreciably higher than that depicted in thedrawings. For example each optical switch may exhibit and extinctionratio of 20 dB or more.

In some embodiments, the extinction ratio of switch 7-105 may not behigh enough to provide a desired turn-off ratio of a pulse. For example,the intensity at the tail 7-150 of a pulse may be too high for someapplications. The inventors have recognized and appreciated that addingattenuating switches 7-120 in an output port may further reduce theintensity of a tail 7-150 at the output port. An attenuating opticalswitch 7-120 may comprise an optical switch of the same type (e.g., aMach-Zender optical switch) that is switched in unison with an upstreamoptical switch 7-105. The attenuating optical switch may have an outputport that is dumped into a beam block 7-110, for example. By addingattenuating optical switches 7-120 to an output port, the extinctionratio of an upstream optical switch (e.g., switch S4) can be increasedas the product of the extinction ratios of the optical switches (S4, S8,S9) that are switched in unison.

The example described in connection with FIG. 7-1B and FIG. 7-1Cutilizes control signal inputs operating at a same frequency for all theoptical switches in the switch array 7-100, but that are staggered intime with respect to one another. In some embodiments, the timing of theswitching control signal may be triggered and/or synchronized from amaster oscillator, e.g., a clock that runs at a frequency that is amultiple of the switching frequency. In some embodiments, differentfrequencies may be applied to the different optical switches along eachoptical path. For example, frequency doubling of a control signal may beimplemented for successive switches along an optical path of the array7-100.

As an example of switching at different frequencies, a first opticalswitch S1 may be driven at a first switching frequency f₁, as depictedin the upper trace of FIG. 7-1D. A second optical switch S2 in theoptical path may be driven at a frequency f₂ that is double the firstfrequency. A third switch S4 in the optical path may be driven at afrequency f₃ that is double the frequency of the second optical switchS2. In some implementations, the drive signals of all the opticalswitches along the optical path may synchronize to the first switch'sdriving signal. The corresponding output pulses from the successiveoutput ports P1, P3, P8, for such an embodiment are depicted in FIG.7-1E. Again, the output pulse is reduced by a factor of two for eachsuccessive switch, although this embodiment requires higher clockfrequencies for successive switches.

One advantage of driving the optical switches 7-105 at differentfrequencies is that the turn-off of a pulse may be increased compared tothe method described above in connection with FIG. 7-1B and FIG. 7-1C.For example and referring to FIG. 7-1E, the tail 7-150 of the outputpulse from an output port P8 may be suppressed by the combined turn-off(product of extinction ratios) of the optical switches S1, S2, and S4 inthe upstream path. This effect can be seen from the loss modulation oftraces in FIG. 7-1D, which shows that each of the switches S1, S2, andS4 are switched to an off state at the tail of the pulse from the outputport P7. Additional attenuating switches 7-120 may, or may not, be addedto the output P7, in some embodiments. A disadvantage of applyingdifferent frequencies to the different optical switches is thathigher-frequency drive signals will be needed for the switching array7-100. For example, a frequency required at the last optical switch maybe on the order of the output pulse duration, in some embodiments.

In some embodiments, a combination of the techniques described inconnection with FIG. 7-1B, FIG. 7-1C and FIG. 7-1D, FIG. 7-1E may beemployed. For example, a first set of optical switches in an opticalpath may be driven with different frequencies as indicated in FIG. 7-1D.Subsequently, a second set of optical switches 7-105 in the same opticalpath may be driven with a same drive frequency, where each drive signalis staggered in time with respect to a preceding drive signal for apreceding optical switch, as indicated in FIG. 7-1B.

III. Coupling Optical Pulses to a Bio-Optoelectronic Chip

According to some implementations, a pulsed laser 1-110 may be mountedin a portable analytical instrument 1-100, and an output of the pulsedlaser may be used to excite biological or chemical samples in one ormore reaction chambers located within the instrument. The instrument mayhave additional optical components between the pulsed laser and reactionchambers arranged to steer an output beam from the pulsed laser to theone or more reaction chambers. As described above, an instrument may beconfigured to receive a bio-optoelectronic chip 1-140 that includes oneor more waveguides and at least one optical coupler (e.g., a gratingcoupler) arranged on the chip to couple optical pulses into the one ormore waveguides. The waveguides may deliver radiation from the opticalpulses to a plurality of reaction chambers, as depicted in FIG. 1-3.Coupling light into an optical waveguide on a chip can require precisealignment of a laser beam to an optical coupler on the chip. In somecases, a beam-steering module may be used to align, in an automatedmanner, a laser beam to an optical coupler on a bio-optoelectronic chip.

An example of a beam-steering module 1-150 is depicted in FIG. 8-1.According to some embodiments, a beam-steering module may comprise asolid chassis 8-110 that is configured to support actuators and opticalcomponents of the beam-steering module. The chassis may be formed orassembled from metal and/or a low-thermal-expansion composite. In somecases, the chassis may be machined or cast from aluminum. The chassis8-110 may be straight or angled (as shown), and may mount to a frame orchassis 1-102 of an instrument in which the pulsed laser 1-110 isincorporated.

The inventors have recognized and appreciated that the beam-steeringmodule's chassis 8-110 can additionally provide support to a PCB 1-130on which a bio-optoelectronic chip 1-140 may be mounted. For example,the chassis 8-110 may attach to the instrument's chassis or frame 1-102at several locations, and a central region of the PCB 1-130 may besecured to the beam-steering module's chassis 8-110 to reduce relativemotion (e.g., motion from mechanical vibrations) between thebeam-steering module and the bio-optoelectronic chip 1-140.

In some embodiments, actuators of a beam-steering module may comprisestepper motors arranged to rotate optical components of thebeam-steering module. To reduce height of the beam-steering module, theactuators may be mounted such that their shafts lie approximately in asame plane, as depicted in the drawing. In some implementations, astepper motor (e.g., as described in U.S. provisional patent application62/289,019) that is fabricated in part on the PCB 1-130, or a separatePCB that mounts to the PCB 1-130, may be used to rotate an opticalcomponent of the beam-steering module about an axis that isperpendicular to the PCB 1-130.

According to some embodiments, a beam-steering module 1-150 may includea first optical flat 8-131, a focusing lens 8-133, a second optical flat8-135, and a third optical flat 8-137. The optical flats and lens may beanti-reflection coated to reduce unwanted Fresnel reflections from theoptics. In some embodiments, there may be a turning mirror 8-134 locatedwithin the beam-steering module, though in some cases a beam paththrough a beam-steering module may be straight and no turning mirror maybe used. According to some implementations, the turning mirror 8-134 maybe dichroic, such that it passes a fundamental wavelength from thepulsed laser 1-110 to a beam dump and/or photodetector and reflects thefrequency-doubled wavelength to the bio-optoelectronic chip 1-140.

The first optical flat 8-131 may be rotated by a first actuator 8-121about an axis that is parallel to the PCB 1-130 to shift the laser beamin an x direction. The second optical flat 8-135 may be rotated by asecond actuator 8-122 about an axis that is perpendicular to the PCB1-130 to shift the laser beam in the y direction. A flexural connection(not shown) may extend from the second actuator 8-122 to the secondoptical flat 8-135 to rotate the second optical flat. The third opticalflat 8-137 may be rotated by a third actuator 8-123 about an axis thatis parallel to the PCB 1-130 to shift the laser beam in an x direction.In some embodiments, there may be a fourth optical flat mounted beforethe lens 8-133 and actuator that is arranged to rotate the fourthoptical flat about an axis that is perpendicular to the PCB 1-130 toshift the laser beam in the z direction. By rotating the optical flats,an optical beam passing through the beam-steering module may betranslated laterally and vertically and its incident angle at the chip1-140 may be changed.

The effects of translating an optical beam in the beam-steering module1-150 can be understood from FIG. 8-2. Translations of the optical beamby rotating optics located after the focusing lens 8-133 results in x, ytranslations at a surface 8-240 (e.g., a surface of a bio-optoelectronicchip) that may be located at a focal point of the lens 8-133. Forexample, a laser beam 8-250 may pass through a focusing lens 8-133 andbe focused onto an optical coupler at the bio-optoelectronic chip 1-140(e.g., focused onto a grating coupler 1-310). Rotation of the secondoptical flat 8-135 about an axis parallel to the y-axis indicated in thedrawing may translate the focused beam at the surface 8-240 in adirection parallel to the x-axis. Rotation of the third optical flat8-137 about an axis parallel to the x-axis may translate the focusedbeam at the surface 8-240 in a direction parallel to the y-axis.

Translations of the optical beam 8-250 by rotating optics located beforethe focusing lens 8-133 results in changing the incident angles of thebeam at the surface 8-240 without appreciably changing the beam's x-ylocation at the surface 8-240. For example, rotation of the firstoptical flat 8-131 about an axis parallel to the y-axis may displace thelaser beam in a direction parallel to the x-axis at the focusing lens8-133. Such movement of the laser beam at the focusing lens will changean incident angle θ_(i) of the laser beam with respect to the z-axis inthe x-z plane at the surface 8-240. In some embodiments, rotation of afourth optical flat 8-132 (not shown in FIG. 8-1) about an axis parallelto the x-axis may change an incident angle ϕ_(i) at the surface 8-240 ina direction lying in the y-z plane. Because the surface 8-240 is locatedat approximately the focal distance f of the lens 8-133, changes inincident angle by translating the beam 8-250 before the lens will notappreciably affect the x-y location of the focused beam at the surface8-240.

In some embodiments, there may be a turning mirror (not shown in FIG.8-2) located between the surface 8-240 of a bio-optoelectronic chip1-140 and the beam-steering module 1-150 to deflect the beam in the −xdirection, so that the chip 1-140 may be oriented with its surface 8-240parallel to the incoming laser beam 8-250. This would allow the chip1-140 to be mounted parallel to an underlying PCB 1-130, as depicted inFIG. 8-1. In some cases, the turning mirror may be formed at low costfrom a small portion (e.g., less than 5 mm square) of a silicon wafer,coated with a reflective material, and mounted within a packagecontaining the bio-optoelectronic chip 1-140.

Referring again to FIG. 1-3 and FIG. 8-1, the x-y position of a laserbeam on a grating coupler 1-310 at a surface of the bio-optoelectronicchip may be adjusted by operating actuators 8-122 and 8-123 to rotateoptical flats 8-135 and 8-137 located after the focusing lens 8-133.When a star coupler or MMI coupler is used to distribute an opticalinput to a plurality of waveguides, the x-y position of the input beamon the grating coupler 1-310 may be adjusted until light couplesapproximately equally to all waveguides connected to the star coupler orMMI coupler. Subsequently, the beam's incident angle θ, in the x-z planemay be adjusted by operating actuator 8-121 to rotate the first opticalflat 8-131. This adjustment may increase an amount of energy coupledinto the waveguide 1-312.

Initially, it was anticipated that changes in a beam's incident angleθ_(i) in the y-z plane (a plane running parallel to the grating teeth ofthe grating coupler 2-310) would not appreciably affect couplingefficiency into the waveguide 1-312. However, the inventors surprisinglydiscovered that changes in this incident angle can have as large aneffect on coupling efficiency as changes in θ_(i). Thelarger-than-expected sensitivity is believed to result from opticalinterference effects between the grating coupler and an underlyingreflective layer (not shown in FIG. 1-3), which is added to increasecoupling efficiency into the waveguide 1-312. According to someembodiments, a beam-steering module may include a fourth optical flat8-132 located before the focusing lens 8-133 that is arranged to affectchanges in the beam's incident angle θ, at the grating coupler.

An advantageous aspect of the beam-steering module 1-150 is thatincident-angle adjustments to θ_(i) and ϕ_(i) may be made substantiallyindependent of x, y adjustments to the position of the focused beam atthe surface 8-240. For example, optical energy from the incident laserbeam 8-250 that is coupled into one or more waveguides 1-312 via agrating coupler 1-310 may be monitored with one or more photodiodes1-324 at an opposite end of the one or more waveguides during analignment procedure that optimizes beam position. Subsequently, beamincident angle may be optimized without appreciably changing the beam'sposition on the grating coupler.

According to some embodiments, an automated alignment procedure may beused to align the laser beam from a pulsed laser 1-110 to a coupler1-310 on a bio-optoelectronic chip 1-140. An alignment procedure maycomprise executing a spiral search for the grating coupler 1-310, asdepicted in FIG. 8-3. The spiral search may be executed by rotating thesecond optical flat 8-135 and the third optical flat 8-137 to translatethe focused beam 8-250 in the x and y directions on the surface of thechip. For example, after a chip 1-140 is loaded into an instrument 1-100and the pulsed laser turned on, the laser beam may strike the surface ofthe chip at the location marked “A” in FIG. 8-3. At this location, theremay be no signal detected by the quad detector 1-320. A spiral searchpath 8-310 may be executed, while signals from the quad detector aremonitored. At location “B” the quad detector may begin to register x, yposition signals of the beam from its detectors. Control circuitry maythen determine the location of the beam with respect to a center of thequad detector, cancel execution of the spiral path, and operate theactuators 8-122 and 8-123 to steer the beam to a center of the quaddetector 1-320, point “C.” The grating coupler 1-310 may be locatedapproximately centrally over the quad detector. Subsequently, fineposition and incident angle adjustments may be made to increase anamount of optical energy coupled into the waveguide 1-312 or waveguides.In some embodiments, the optical powers from multiple integratedphotodiodes 1-324 at the ends of multiple waveguides 1-312 aremonitored, so that fine adjustments may be made to the laser beam at thegrating coupler to increase uniformity of the powers coupled into themultiple optical waveguides.

Other methods and apparatus may be used to search for the quad detector1-320 and align the focused beam 8-250 to the grating coupler 1-310. Insome embodiments, the sensitivity of the quad detector 1-320 can beimproved to expand the range over which the laser beam may be detected.For example, signals from the quad detector with the laser power at ahigh power (e.g., fully on) may be compared against signals from thequad detector with the laser power at a low setting (e.g., off).Additionally, the signals may be integrated over longer periods of timeto improve the location-detection sensitivity of the quad detector, whenthe laser beam may be located at an appreciable distance from the quaddetector.

In some embodiments, light scattering elements (not shown in FIG. 8-3)may be fabricated on the chip 1-140 around the quad detector 1-320. Whenthe focused beam is misaligned and at a peripheral location away fromthe quad detector, the scattering elements may scatter light from thefocused beam towards the quad detector 1-320. The detected scatteredlight may then indicate a position of the beam.

In some implementations, a narrow, linear scattering element or linedetector, similar in width to the anticipated focused beam size, may beplaced through the center of the quad detector (or in any suitableorientation with respect to the quad detector), and extend significantlybeyond opposite edges of the quad detector (e.g., to a distance greaterthan a reasonable expectation of initial beam offset error). Since theorientation of this element or detector is known by design, the focusedbeam 8-250 can first be scanned in a direction perpendicular to theelement until the beam strikes the element or detector and is positivelydetected, either by scatter to the quad detector 1-320, or directly bythe line detector. Then, the beam may be scanned in the other directionto find the quad detector 1-320.

According to some embodiments, the laser beam may be initially expandedat the surface 8-240 of the chip 1-140 (e.g., defocusing the beam bymoving lens 8-133 with an actuator or using other means). The footprintof the beam on the chip may then be greatly increased (e.g., by a factorof 10 or more) so that any scanning process can use larger steps betweenbeam positions when searching for the quad detector 1-320 (e.g., largeroffsets between radial loops on a spiral scan). This and the foregoingalternative searching methods may reduce the search time associated withaligning the focused beam 8-250 to the grating coupler 1-310.

After alignment, the incident laser beam may be maintained actively inan aligned position. For example, an x, y position of the beamdetermined after the initial alignment with respect to the quad detector1-320 may be actively maintained using feedback from the quad detectorand activation of the actuators 8-122 and 8-123 to maintain the beam inan approximately fixed location. In some embodiments, incident angles ofthe optical beam at the surface may not be adjusted after an initialalignment to optimize power coupled into the waveguide. Additionally, anamount of power coupled into the waveguides may be maintained atapproximately a constant level throughout measurements.

Power delivered to the waveguides may be maintained at approximatelyconstant levels by monitoring photodiode 1-324 signals from oppositeends of the waveguides and feeding that signal to a controller thatoperates an actuator 2-162 that controls an orientation of a half-waveplate 2-160 of the pulsed laser system 1-110 (referring to FIG. 2-1A).Rotation of the half wave plate 2-160 changes the polarization of theoptical pulses entering the frequency-doubling crystal 2-170, andtherefor changes the conversion efficiency to the shorter wavelengthused to excite fluorophores in the reaction chambers.

Example circuitry for beam alignment and power stabilization is depictedin FIG. 8-4, according to some embodiments. The quad detector 1-320 isrepresented as four photodiodes, and a waveguide photodiode 1-324 isrepresented as a fifth photodiode. In some implementations, there may bea large plurality of waveguides to which optical power is coupled from asingle grating coupler. Accordingly, there may be a large plurality ofwaveguide photodiodes 1-324 at end of the waveguide that have signaloutputs connected to control circuitry 8-430. Amplifying circuitry 8-410may be arranged to detect voltages produced by photoconduction of thediodes. The amplifying circuitry 8-410 may comprise CMOS electronics(e.g., FETs, sampling circuits, analog-to-digital converters) thatconvert an analog signal to a digital signal, according to someembodiments. In other embodiments, analog signals may be provided fromthe amplifying circuitry to control circuitry 8-430.

In some embodiments, control circuitry may comprise one or a combinationof the following elements: analog and digital circuitry, an ASIC, anFPGA, a DSP, a microcontroller, and a microprocessor. The controlcircuitry 8-430 may be configured to process received signals from theone or more waveguide photodiodes to determine a level of optical powerin each waveguide. Control circuitry 8-430 may be further configured toprocess received signals from the quad detector 1-320 to determine an x,y location of the optical beam with respect to the quad detector. Insome implementations, the control circuitry 8-430 is configured todetect power coupled into each waveguide, and provide a control signalto the actuators to move the laser beam such that power is equalized inthe waveguides or has a highest uniformity across the waveguides.

A position of the laser beam in the x direction may be determined, forexample, by control circuitry 8-430 adapted to execute the followingalgorithm:

S _(x)=[(V _(Q2) +V _(Q3))−(V _(Q1) +V _(Q4))]/V _(T)

where S_(x) is a normalized signal level corresponding to the xdirection, V_(Qn) is a signal level (e.g., voltage) received from then^(th) photodiode of the quad detector, and V_(T) is a total signallevel received by summing the signal from all four photodiodes.Additionally, a position of the laser beam in the y direction may bedetermined, for example, using the following algorithm:

S _(y)=[(V _(Q3) +V _(Q4))−(V _(Q1) +V _(Q2))]/V _(T).

An average power coupled into all waveguides on the chip 1-140 may bedetermined by summing signals from all of the photodiodes 1-324 arrangedto detect power in each of the waveguides on the chip.

Control signals may be generated by control circuitry 8-430 responsiveto detected beam position in x and y and responsive to power levelsdetected in one or more waveguides of the bio-optoelectronic chip 1-140.The control signals may be provided as digital signals overcommunication links (SM1, SM2, SM3) to actuators of the beam-steeringmodule 1-150 and a communication link WP to an actuator 1-162 of thepulsed laser system 1-110 that controls rotation of the half-wave plate2-160.

To further illustrate operation of the pulsed laser 1-110 and instrument1-100, an example method 8-500 for aligning and maintaining alignment ofthe pulsed-laser beam to an optical coupler (e.g., a grating coupler) ona bio-optoelectronic chip 1-140 is illustrated in FIG. 8-5. According tosome embodiments, control circuitry 8-430 within instrument 1-100 may beconfigured to detect (act 8-505) the loading of a bio-optoelectronicchip in the instrument. When a new chip is loaded, its optical couplermay not be aligned to the laser beam from the pulsed laser. Responsiveto detection of the loading, control circuitry 8-430 may operate thebeam-steering module 1-150 to execute (act 8-510) spiral scanning (orany other suitable scanning method described above) of the pulsed-laserbeam over the surface of the bio-optoelectronic chip, as depicted inFIG. 8-3, for example. The control circuitry may operate actuators8-122, 8-123 of the beam-steering module 1-150 to move the beam in aspiral path 8-310, or any other suitable path. While the pulsed-laserbeam is being scanned over the surface of the chip, signals from a quaddetector 1-320 may be monitored (act 8-515) by control circuitry 8-430to determine whether a position of the laser beam is detected.

If signals from the quad detector indicate (act 8-520) that a positionof the pulsed-laser beam has not been detected, then the controlcircuitry may continue scanning (act 8-510) the laser beam over thesurface of the bio-optoelectronic chip. Alternatively, if the beam'sposition has been detected, the spiral scan may be stopped and theactuators of the beam-steering module may be driven to approximatelycenter (act 8-525) the pulsed-laser beam over the quad detector 1-320.According to some embodiments, a grating coupler 1-310 may beapproximately centered over the quad detector, so that centering thelaser beam over the quad detector approximately aligns the beam to thegrating coupler. With the pulsed-laser beam at the approximate locationof the grating coupler, the control circuitry may drive actuators 8-122,8-123 of the beam-steering module 1-150 to execute (act 8-530) an x-yscan in the immediate vicinity of the grating coupler. For example, thebeam-steering module may execute a sequential linear scan in the xdirection to find a first optimum coupling value and then a linear scanin the y direction to find a second optimum coupling value. While thelaser beam is being scanned, output signals from the quad detector 1-320and one or more waveguide photodiodes 1-324 may be monitored (act8-535).

As the pulsed-laser beam is scanned in the vicinity of the gratingcoupler, power detected from the one or more waveguide photodiodes 1-324may increase and decrease. In some embodiments, there may be a maximumin total power coupled into the waveguides (detected by one or morewaveguide photodiodes 1-324) corresponding to a first x₁, y₁ position ofthe pulsed-laser beam (as determined by the quad detector 1-320). Insome cases, there may be a second x₂, y₂ position of the pulsed-laserbeam for which power levels detected in a plurality of waveguidesconnected to the grating coupler are approximately equal (e.g., within±20% or even within ±10%). At the second position, the total powercoupled into the waveguides may be less than the amount coupled into thewaveguides in the first position.

According to some embodiments, control circuitry 8-430 may be adapted tomove the pulsed-laser beam until a highest total power coupled into thewaveguides within a predetermined uniformity (e.g., ±15%) acrosswaveguides is achieved. The corresponding location may be a firstoptimized location x₃, y₃, which may be different from the firstposition x₁, y₁ and second position x₂, y₂. In some implementations,larger power variations across waveguides may be tolerated (e.g.,normalized out of the resulting data). In such implementations, thefirst optimized location x₃, y₃ may be a location at which total powerinto the waveguides is maximized.

If control circuitry 8-430 determines (act 8-540) that a first optimizedlocation x₃, y₃ has not been found, control circuitry may continueoperating the actuators of the beam-steering module to execute (act8-530) an x-y scan of the pulsed-laser beam in the vicinity of thegrating coupler 1-310. If a first optimized coupling location has beenfound, then control circuitry 8-430 may hold (act 8-545) thelaser-beam's position by operating actuators 8-122 and 8-123 to maintainthe laser beam at a fixed location sensed by the quad detector 1-320.Control circuitry may then actuate actuator 8-121 and optionally anadditional actuator of the beam-steering module to scan (act 8-550)incident beam angles at the optical coupler on the bio-optoelectronicchip. As the incident beam angles are being scanned, signal levels fromwaveguide photodiodes 1-324 in one or more waveguides may be monitored(act 8-555). The incident beam angles may be scanned until controlcircuitry 8-430 determines (act 8-560) that a second optimized couplingorientation has been found. The second optimized coupling orientationmay correspond to beam incidence angles that provide a highest amount ofpower coupled into one or more waveguides on the bio-optoelectronic chip1-140, or a highest power coupled into the waveguides with apredetermined uniformity of power across the waveguides.

If a second optimized coupling orientation has not been identified (act8-560), then control circuitry may continue the scan (act 8-550) ofincident beam angles. If the second optimize coupling orientation hasbeen identified, then the control circuitry 8-430 may maintain (act8-565) the pulsed-laser beam's x-y position as well as its incidentangles. With the pulsed-laser beam's position and incident anglesmaintained, a measurement on the bio-optoelectronic chip 1-140 maybegin.

In some embodiments, the pulsed-laser beam's position may be maintainedwith respect to the quad detector 1-320 during a measurement, whichcould last for 10's of minutes or longer. For example, active feedbackmay be employed to sense the beam's position at the optical coupler(with quad detector 1-320) and maintain the pulsed laser beam at thesensed position (for example, by operating actuators 8-122 and 8-123 tocompensate for drift or vibrations in the system).

As a measurement commences, optical power levels in the reactionchambers may also be maintained (act 8-570). According to someembodiments, maintaining the optical power level may comprise monitoringwaveguide power levels with one or more waveguide photodiodes 1-324located at the end of one or more waveguides, and compensating forchanges in optical power by actuating actuator 2-162 of the pulsed lasersystem 1-110. Operation of the actuator will rotate the half-wave plate2-160, which rotates the optical polarization in the frequency-doublingcrystal 2-170 and changes conversion efficiency to the frequency-doubledwavelength. In this manner, power fluctuations that would otherwiseoccur in the reaction chambers can be significantly reduced.

In some embodiments, control circuitry 8-430 may receive anend-of-measurement signal from the bio-optoelectronic chip 1-140 at theconclusion of a measurement. If the control circuitry does not detect(act 8-575) an end-of-measurement signal, the beam orientation and powerlevels may be maintained. If the control circuitry detects (act 8-575)an end-of-measurement signal, the process may end. In some embodiments,ending the process may comprise powering down the pulsed laser 1-110 andactuators of the beam-steering module.

IV. Clock Generation and System Synchronization

Referring again to FIG. 1-1, regardless of the method and apparatus thatis used to produce short or ultrashort-pulses, a system 1-100 mayinclude circuitry configured to synchronize at least some electronicoperations (e.g., data acquisition and signal processing) of an analyticsystem 1-160 with the repetition rate of optical pulses 1-122 from theoptical source 1-110. There are at least two ways to synchronize thepulse repetition rate to electronics on the analytic system 1-160.According to a first technique, a master clock may be used as a timingsource to trigger both generation of pulses at the pulsed optical sourceand instrument electronics. In a second technique, a timing signal maybe derived from the pulsed optical source and used to trigger instrumentelectronics.

FIG. 9-1 depicts a system in which a clock 9-110 provides a timingsignal at a synchronizing frequency f_(sync) to both a pulsed opticalsource 1-110 (e.g., a gain-switched pulsed laser or LED) and to ananalytic system 1-160 that may be configured to detect and processsignals that result from interactions between each excitation pulse1-120 and biological, chemical, or other physical matter. As just oneexample, each excitation pulse may excite one or more fluorescentmolecules of a biological sample that are used to analyze a property ofthe biological sample (e.g., nucleotide incorporation for DNAsequencing, cancerous or non-cancerous, viral or bacterial infection,blood glucose level). For example, non-cancerous cells may exhibit acharacteristic fluorescent lifetime of a first value τ₁, whereascancerous cells may exhibit a lifetime of a second value τ₂ that isdifferent from and can be distinguished from the first lifetime value.As another example, a fluorescent lifetime detected from a sample ofblood may have a lifetime value and/or intensity value (relative toanother stable marker) that is dependent on blood glucose level. Aftereach pulse or a sequence of several pulses, the analytic system 1-160may detect and process fluorescent signals to determine a property ofthe sample. In some embodiments, the analytic system may produce animage of an area probed by the excitation pulses that comprises a two orthree-dimensional map of the area indicating one or more properties ofregions within the imaged area.

Regardless of the type of analysis being done, detection and processingelectronics on the analytic system 1-160 may need to be carefullysynchronized with the arrival of each optical excitation pulse. Forexample, when evaluating fluorescent lifetime, it is beneficial to knowthe time of excitation of a sample accurately, so that timing ofemission events can be correctly recorded.

A synchronizing arrangement depicted in FIG. 9-1 may be suitable forsystems in which the optical pulses are produced by active methods(e.g., external control). Active pulsed systems may include, but are notlimited to gain-switched lasers and pulsed LEDs. In such systems, aclock 9-110 may provide a digital clock signal that is used to triggerpulse production (e.g., gain switching or current injection into an LEDjunction) in the pulsed optical source 1-110. The same clock may alsoprovide the same or synchronized digital signal to an analytic system1-160, so that electronic operations on the instrument can besynchronized to the pulse-arrival times at the instrument.

The clock 9-110 may be any suitable clocking device. In someembodiments, the clock may comprise a crystal oscillator or a MEMS-basedoscillator. In some implementations, the clock may comprise a transistorring oscillator.

The frequency f_(sync) of a clock signal provided by the clock 9-110need not be a same frequency as the pulse repetition rate R. The pulserepetition rate may be given by R=1/T, where T is the pulse-separationinterval. In FIG. 9-1, the optical pulses 1-120 are depicted as beingspatially separated by a distance D. This separation distancecorresponds to the time T between arrival of pulses at the analyticsystem 1-160 according to the relation T=D/c where c is the speed oflight. In practice, the time T between pulses can be determined with aphotodiode and oscilloscope. According to some embodiments, T=f_(sync)/Nwhere N is an integer greater than or equal to 1. In someimplementations, T=Nf_(sync) where N is an integer greater than or equalto 1.

FIG. 9-2 depicts a system in which a timer 9-220 provides asynchronizing signal to the analytic system 1-160. In some embodiments,the timer 9-220 may derive a synchronizing signal from the pulsedoptical source 1-110, and the derived signal is used to provide asynchronizing signal to the analytic system 1-160.

According to some embodiments, the timer 9-220 may receive an analog ordigitized signal from a photodiode that detects optical pulses from thepulse source 1-110. The timer 9-220 may use any suitable method to formor trigger a synchronizing signal from the received analog or digitizedsignal. For example, the timer may use a Schmitt trigger or comparatorto form a train of digital pulses from detected optical pulses. In someimplementations, the timer 9-220 may further use a delay-locked loop orphase-locked loop to synchronize a stable clock signal to a train ofdigital pulses produced from the detected optical pulses. The train ofdigital pulses or the locked stable clock signal may be provided to theanalytic system 1-160 to synchronize electronics on the instrument withthe optical pulses.

The inventors have recognized and appreciated that coordination ofoperation of the pulsed laser 1-110 (e.g., to deliver excitation opticalpulses to reaction chambers 1-330), signal-acquisition electronics(e.g., operation of time-binning photodetectors 1-322), and dataread-out from the bio-optoelectronic chip 1-140 poses technicalchallenges. For example, in order for the time-binned signals collectedat the reaction chambers to be accurate representations of fluorescentdecay characteristics, each of the time-binning photodetector 1-322 mustbe triggered with precise timing after the arrival of each excitationoptical pulse at the reaction chambers. Additionally, data must be readfrom the bio-optoelectronic chip 1-140 in approximate synchronicity withdata acquisition at the reaction chambers to avoid data overruns andmissed data. Missed data could be detrimental in some cases, e.g.,causing a misrecognition of a gene sequence. The inventors haverecognized and appreciated that system timing is further complicated bythe natural operating characteristics of passively mode-locked lasers,e.g., prone to fluctuations in pulse amplitude, fluctuations inpulse-to-pulse interval T, and occasional pulse drop-outs.

The inventors have conceived and developed clock-generation circuitrythat may be used to generate a clock signal and drive data-acquisitionelectronics in a portable instrument 1-100. An example ofclock-generation circuitry 9-300 is depicted in FIG. 9-3. According tosome embodiments, clock-generation circuitry may include stages of pulsedetection, signal amplification with automatic gain control, clockdigitization, and clock phase locking.

A pulse-detection stage may comprise a high-speed photodiode 9-310 thatis reversed biased and connected between a biasing potential and areference potential (e.g., a ground potential), according to someembodiments. A reverse bias on the photodiode may be any suitable value,and may be fixed using fixed-value resistors or may be adjustable. Insome cases, a capacitor C may be connected between a cathode of thephotodiode 9-310 and a reference potential. A signal from the anode ofthe photodiode may be provided to an amplification stage. In someembodiments, the pulse detection stage may be configured to detectoptical pulses having an average power level between about 100microwatts and about 25 milliwatts. The pulse-detection stage of theclock-generation circuitry 9-300 may be mounted on or near the pulsedlaser 1-110, and arranged to detect optical pulses from the laser.

An amplification stage may comprise one or more analog amplifiers 9-320that may include variable gain adjustments or adjustable attenuation, sothat pulse output levels from the analog gain amplifiers may be setwithin a predetermined range. An amplification stage of theclock-generation circuitry 9-300 may further include an automatic gaincontrol amplifier 9-340. In some cases, analog filtering circuitry 9-330may be connected to an output of the analog amplifiers 9-320 (e.g., toremove high-frequency (e.g., greater than about 500 MHz) and/orlow-frequency noise (e.g., less than about 100 Hz)). The filtered orunfiltered output from the one or more analog gain amplifiers 9-320 maybe provided to an automatic gain control amplifier 9-340, according tosome embodiments.

According to some embodiments, a final output signal from the one ormore analog amplifiers may be positive-going. The inventors haverecognized and appreciated that a subsequent automatic gain-control(AGC) amplifier operates more reliably when it input pulses to positivevoltage rather than negative voltage. The automatic gain controlamplifier may vary its internal gain to compensate for amplitudefluctuations in the received electronic pulse train. The output pulsetrain from the automatic gain control amplifier 9-340 may haveapproximately constant amplitude, as depicted in the drawing, whereasthe input to the automatic gain control amplifier 9-340 may havefluctuations in the pulse-to-pulse amplitudes. An example automatic gaincontrol amplifier is model AD8368 available from Analog Devices, Inc. ofNorwood, Mass.

In a clock digitization stage, an output from the automatic gain controlamplifier may be provided to a comparator 9-350 to produce a digitalpulse train, according to some implementations. For example, the pulsetrain from the AGC may be provided to a first input of the comparator9-350, and a reference potential (which may be user-settable in someembodiments) may be connected to a second input of the comparator. Thereference potential may establish the trigger point for the rising edgeof each produced digital pulse.

As may be appreciated, fluctuations in optical pulse amplitude wouldlead to fluctuations in amplitudes of the electronic pulses before theAGC amplifier 9-340. Without the AGC amplifier, these amplitudefluctuations would lead to timing jitter in the rising edges of pulsesin the digitized pulse train from the comparator 9-350. By leveling thepulse amplitudes with the AGC amplifier, pulse jitter after thecomparator is reduced significantly. For example, timing jitter can bereduced to less than about 50 picoseconds with the AGC amplifier. Insome implementations, an output from the comparator may be provided tologic circuitry 9-370 which is configured to change the duty cycle ofthe digitized pulse train to approximately 50%.

A phase-locking stage of the clock-generation circuitry 9-300 maycomprise a phase-locked loop (PLL) circuit 9-380 that is used to produceone or more stable output clock signals for timing and synchronizinginstrument operations. According to some embodiments, an output from theclock digitization stage may be provided to a first input (e.g., afeedback input) of a PLL circuit 9-380, and a signal from an electronicor electro-mechanical oscillator 9-360 may be provided to a second input(e.g., a reference input) to the PLL. An electronic orelectro-mechanical oscillator may be highly stable against mechanicalperturbations and against temperature variations in some cases.According to some embodiments, a phase of the stable clock signal fromthe electronic or electro-mechanical oscillator 9-360 is locked, by thePLL, to a phase of the digitized clock signal derived from themode-locked laser, which may be less stable. In this manner, theelectronic or electro-mechanical oscillator 9-360 can ride throughshort-term instabilities (e.g., pulse jitter, pulse drop outs) of thepulsed laser 1-110, and yet be approximately synchronized to the opticalpulse train. The phase-locked loop circuit 9-380 may be configured toproduce one or more stable output clock signals that are derived fromthe phase-locked signal from the electro or electro-mechanicaloscillator 9-360. An example circuit that may be used to implement thePLL is IC chip Si5338, which is available from Silicon Laboratories Inc.of Austin, Tex.

According to some embodiments, one or more clock signals output from thePLL circuit 9-380 may be provided to the bio-optoelectronic chip 1-140to time data-acquisition electronics on the chip. In some cases, the PLLcircuit 9-380 may include phase adjustment circuitry 9-382, 9-384 on itsclock outputs, or separate phase adjustment circuits may be connected toclock outputs of the phase-locked loop. In some implementations, thebio-optoelectronic chip 1-140 may provide a pulse-arrival signal 1-142from one or more photodetectors on the chip that indicate the arrival ofoptical excitation pulses from the pulsed laser 1-110. The pulse-arrivalsignal may be evaluated and used to set the phase or phases of clocksignals provided to the bio-optoelectronic chip 1-140. In someembodiments, the pulse-arrival signal may be provided back to thephased-locked loop circuit 9-380 and processed to automatically adjustthe phase of the clock signal(s) provided to the chip, so that a triggeredge of a clock signal provided to drive data-acquisition on thebio-optoelectronic chip 1-140 (e.g., timing of signal acquisition by thetime-binning photodetectors 1-322) is adjusted to occur at apredetermined time after the arrival of an optical excitation pulse inthe reaction chambers.

According to some embodiments, a clock signal from the PLL circuit 9-380may also be provided to one or more field-programmable gate arrays(FPGAs) 9-390 included in the instrument 1-100. The FPGAs may be usedfor various functions on the instrument, such as driving data read outfrom the bio-optoelectronic chip 1-140, data processing, datatransmission, data storage, etc.

The inventors have recognized and appreciated that there can be aninterplay between the loop bandwidth of the AGC amplifier 9-340 and theloop bandwidth of the phase-locked loop 9-390. For example, if the loopbandwidth of the phase-locked loop is too high, the PLL may respond tojitter introduced by the AGC amplifier and comparator in the digitizedpulse train, and not accurately track the optical pulse timing. On theother hand, if either or both of the AGC and PLL loop bandwidths are toolow, the resulting clock signals output from the PLL will not accuratelytrack the optical pulse timing. The inventors have found that anintegration time constant associated with the loop bandwidth of the PLL9-390 should be between about 30 pulses and about 80 pulses of theoptical pulse train from the mode-locked laser 1-110. Additionally, anintegration time constant associated with the loop bandwidth of the AGCamplifier 9-340 should not exceed by more than about 20% the integrationtime constant for the PLL.

In some implementations, one or more signals from the amplificationstage may be used for additional purposes in the instrument 1-100. Forexample, an analog signal 9-332 may be split off prior to the AGCamplifier 9-340 and used to monitor the quality of mode locking in thepulsed laser 1-110. For example, the analog signal 9-332 may be analyzedelectronically in the frequency and/or time domain to detectcharacteristics that are indicative of the onset of Q-switching by thepulsed laser. If the characteristics (and onset of Q-switching) aredetected, the system may automatically make adjustments to optics withinthe mode-locked laser (e.g., cavity-alignment optics) to avoidQ-switching, or the system may indicate an error and/or shut down thepulsed laser.

In some embodiments, an AGC amplifier may provide an output signal 9-342(analog or digital) that is representative of real-time gain adjustmentsthat are needed to level the amplitudes of the output pulses. Theinventors have recognized and appreciated that this output signal 9-342may be used to evaluate mode-locking quality of the pulsed laser. Forexample, its spectrum may be analyzed to detect the onset ofQ-switching.

Although clock generation and synchronization has been described usingan automatic gain control amplifier and a phase-locked loop, alternativeapparatus may be used in other embodiments for which a larger amount ofclock jitter (e.g., up to about 300 ps) may be tolerated. In someimplementations, an amplifier in the pulse amplification stage may bedriven into saturation to provide a rising edge trigger signal. Atrigger point for a clock may be set at some value on the rising edge.Because the amplifier saturates, variations in pulse amplitude have lessof an effect on the trigger timing than for a non-saturated amplifier.The rising edge may be used to toggle a flip-flop clocking circuit, suchas those implemented in field-programmable gate arrays (FPGAs). Thefalling edge from the saturated amplifier returning back to zero canhave appreciably more timing variability, depending on when the outputof the amplifier is released from saturation. However, the falling edgeis not detected by the flip-flop clocking circuit and has no effect onthe clocking.

Many FPGAs include digital delay-lock loops (DLL) which may be usedinstead of a PLL to lock a stable oscillator to the laser-generatedclocking signal from the flip flop. In some embodiments, the receivingflip-flop divides the clocking rate from the optical pulse train by two,which can provide a 50% duty-cycle clock signal to the DLL at one-halfthe pulse repetition rate. The DLL may be configured to generate afrequency-doubled clock to be synchronized with the optical pulse train.Additional synchronized, higher-frequency clocks may also be generatedby the DLL and FPGA.

In some embodiments, two or more pulsed optical sources 1-110 a, 1-110 bmay be needed to supply optical pulses at two or more differentwavelengths to an analytic system 1-160, as depicted in FIG. 9-4. Insuch embodiments, it may be necessary to synchronize pulse repetitionrates of the optical sources and electronic operations on the analyticsystem 1-160. In some implementations, if two pulsed optical sources useactive methods (e.g., gain switching) to produce pulses, the techniquesdescribed above in connection with FIG. 9-1 may be used. For example, aclock 9-110 may supply a clock or synchronizing signal at asynchronizing frequency f_(sync) to drive circuits for both pulsedoptical sources 1-110 a, 1-110 b, and to the analytic system 1-160. Ifone optical pulse source 1-110 b produces pulses using passive methods,then the techniques described in connection with FIG. 9-2 may be used toderive a synchronizing signal from the passive pulse source. Thesynchronizing signal may then be provided to the active pulse source1-110 a to synchronize pulse production by that source and to theinstrument 1-160 to synchronize instrument electronics and operations.

When pulses are produced actively at each optical source, it may or maynot be necessary to dynamically adjust a laser cavity length using afeedback-control system for stable and synchronized pulse production. Ifpulses are produced by gain switching of a laser's gain medium, thenlaser cavity length adjustment may not be needed. If pulses are producedby active mode-locking techniques, then a dynamic laser cavity lengthadjustment may be needed to produce a stable train of optical pulses.There are several electro-mechanical techniques by which laser cavitylength adjustments may be made. For example, a cavity mirror (such as acavity end mirror or turning mirror pair) may be positioned using apiezoelectric transducer that is controlled according to a feedbacksignal. The feedback signal may be derived from a difference between apulse repetition rate produced by the laser cavity and another pulserepetition rate or clock signal produced externally. In some cases, afiber laser length may be stretched using a piezoelectric materialaccording to a feedback signal. In some implementations, a cavity mirrormay be a microelectromechanical-based mirror that is controlledaccording to a feedback signal.

According to some embodiments, two optical pulse sources 1-110 a, 1-110b may both produce optical pulses passively (e.g., by passive modelocking). In such embodiments, a synchronizing signal may be derivedfrom one of the pulsed optical sources, as described in connection withFIG. 9-3, for inter-laser pulse and electronic synchronization.Additional measures may be needed to synchronize pulses from the secondoptical pulse source to pulses from the first optical source. Forexample, a timing signal may also be derived from the second opticalpulse source, and used with an electro-mechanical feedback circuit tocontrol a cavity length of the second optical pulse source. Bycontrolling the cavity length of the second optical pulse source, thetiming signal derived from the second optical pulse source can be lockedin frequency and phase (e.g., via a phase-locked loop) to a clock signalderived from the first optical pulse source. In this manner, a pulsetrain from a second optical pulse source can be synchronized to a pulsetrain of the first optical pulse source, and instrument operations andelectronics may also be synchronized to the first optical pulse source.

In some implementations, it may be beneficial to interleave pulses intime from two pulsed optical sources, as depicted in FIG. 9-5A and FIG.9-5B. When pulses are interleaved, a pulse 9-120 a from a first source1-110 a may excite one or more samples at the analytic system 1-160 witha first characteristic wavelength λ₁ at a first time t₁. Datarepresentative of the first pulse's interaction with the one or moresamples may then be collected by the instrument. At a later time t₂, apulse 9-120 b from a second source 1-110 b may excite one or moresamples at the analytic system 1-160 with a second characteristicwavelength λ₂. Data representative of the second pulse's interactionwith the one or more samples may then be collected by the instrument. Byinterleaving the pulses, effects of pulse-sample interactions at onewavelength may not intermix with effects of pulse-sample interactions ata second wavelength. Further, characteristics associated with two ormore fluorescent markers may be detected.

Pulses may be interleaved with timing and synchronization circuitry, asdepicted in FIG. 9-5A. Methods described in connection with FIG. 9-4 maybe used to synchronize pulse trains from the two pulsed optical sources1-110 a, 1-110 b, and to synchronize electronics and operations on theanalytic system 1-160 with the arrival of pulses. To interleave thepulses, pulses of one pulsed optical source may be phase-locked ortriggered out of phase with pulses from the other pulsed optical source.For example, pulses of a first pulsed optical source 1-110 a may bephase-locked (using a phase-locked loop or delay-locked loop) ortriggered to be 180 degrees out of phase with pulses from the secondpulsed optical source 1-110 b, though other phase or angle relationshipsmay be used in some embodiments. In some implementations, a timing delaymay be added to a trigger signal provided to one of the pulsed opticalsources. The timing delay may delay a trigger edge by approximatelyone-half the pulse-separation interval T. According to some embodiments,a frequency-doubled synchronization signal may be generated by a timer9-220, and provided to the instrument 9-160 for synchronizing instrumentelectronics and operations with the arrival of interleaved pulses fromthe pulsed optical sources.

The inventors have conceived further methods and techniques by whichoptical pulse trains at two or more different characteristic wavelengthscan be produced and synchronized. FIG. 9-6A depicts a two-laser system9-600 that employs nonlinear optical material to generate twosynchronized pulse trains 9-120 c, 9-120 d at desired characteristicwavelengths λ₁/2 and λ₃. According to some embodiments, a first laser1-110 a may produce a first train of optical pulses 9-120 a at a firstcharacteristic wavelength λ₁. For example, the first laser may be apassively mode-locked laser (e.g., a Nd:YVO₄ or Nd:GdVO₄ laser) thatproduces pulses at 1064 nm. The first laser 1-110 a may comprise anylaser cavity system described in connection with FIG. 3-3A or FIG. 5-1through FIG. 5-3. The first train of optical pulses 9-120 a may befrequency doubled by second harmonic generation (SHG) in a firstnonlinear optical element 9-610 (e.g., a KTP or BBO crystal) to producea third train of optical pulses 9-120 c at one-half the wavelength(e.g., λ₁/2=532 nm) of the first laser's pulse train. The secondharmonic generation will not convert all of the pulse energy to thesecond harmonic frequency, so that an attenuated pulse train at thefundamental wavelength λ₁ will emerge from the first nonlinear opticalelement 9-610.

Additionally, a second passively mode-locked laser 1-110 b may produce asecond train of optical pulses 9-120 b at a second characteristicwavelength λ₂. In some embodiments, the second laser may also comprise apassively mode-locked laser (e.g., a Nd:YVO₄ or Nd:GdVO₄ laser) thatproduces pulses at a second wavelength (e.g., 1342 nm) that is a secondlasing transition supported by the same type of gain medium, althoughother lasing materials may be used in other embodiments. A firstdichroic mirror DC₁ may be used to direct pulses from the first laser1-110 a to a second dichroic mirror DC₂ where pulse trains from the twolasers will be combined and directed to a second nonlinear opticalelement 9-620 (e.g., a KTP or BBO crystal). In the second nonlinearelement, the optical fields from the two pulse trains interact, providedthe pulses arrive together, to generate a third wavelength λ₃ by aprocess known as sum-frequency generation (SFG). In this process, theresulting wavelength is given by the following relation.

λ₃=λ₁λ₂/(|λ₁+λ₂|)  (2)

According to the example above, the third wavelength λ₃ may be producedat about 593.5 nm. As a result, the two-laser system can produce a thirdpulse train 9-120 c at 532 nm and a fourth pulse train 9-120 d at 593.5nm. In some embodiments, the fourth pulse train may contain pulses atthe fundamental wavelengths λ₁ and λ₂, but this radiation can befiltered out of the pulse train using an infrared filter, for example.

In some implementations, a fifth pulse train at a fourth characteristicwavelength λ₄ (not shown) may be produced. For example, radiation fromthe second laser 1-110 b at its fundamental wavelength λ₂ may emergefrom the second nonlinear optical element 9-620 and be frequency doubledin a third nonlinear optical element (not shown). According to theexample above, the fourth characteristic wavelength would be about 670nm. Additionally, these pulses would be synchronized in time with theother optical pulses in the third and fourth pulse trains.

As noted above, the pulses from the two lasers 1-110 a, 1-110 b shouldarrive at the second nonlinear optical element 9-620 at a same time andbe spatially overlapped as much as possible in the element. Accordingly,the two lasers should be synchronized. Synchronization of the twolasers, and to instrument electronics, may be done using a timing andelectro-mechanical control circuit 9-220, as described in connectionwith FIG. 9-4, for example. The control circuit 9-220 may, in someembodiments, compare the pulse repetition rates from the two lasers toproduce a feedback signal that is used to control a cavity length of onelaser. A cavity length may be controlled via an electro-mechanicalactuator, such as a piezoelectric transducer. The control circuit 9-220may further generate, or phase lock to, a clocking signal that is usedto synchronize electronic operations of an instrument 1-160 whichanalyzes interactions of the pulses with matter.

Synchronized pulses at multiple characteristic wavelengths generated bythe lasing system of FIG. 9-6A, or other lasing systems or combinationsof lasing systems described herein, may be desirable for excitingfluorophores for bioanalytical systems. In some implementations, signalsrepresentative of optical emission detected from excited fluorophoresmay be processed to distinguish the type of fluorophore, according tomethods described in the related applications. In some cases, theanalysis of the detected signals may distinguish the fluorophores basedon their lifetimes and/or spectral characteristic. In some embodiments,distinguishing fluorophores based on lifetime favors the use of multipleexcitation wavelengths at the sample, because the different fluorophoresto be distinguished may have different absorption bands. Excitationpulses at multiple wavelengths can assure that each fluorophore, whenpresent at the sample, will be excited.

In some cases, when multiple excitation wavelengths are available,fluorophores may be distinguished based on whether or not an excitationsource excites a fluorophore. As just one example, four fluorophores maybe used in a single-molecule gene-sequencing apparatus to detectnucleotide incorporation into a gene or gene fragment. The fourfluorophores may be selected, such that they have reduced overlap intheir absorption bands. Four excitation wavelengths, matched to theabsorption bands, from two or more pulsed laser sources may be used toexcite the fluorophores. The pulses may be interleaved in time, so thatpulses arrive at a sample within different time intervals for eachcharacteristic wavelength. If a fluorophore having an absorption bandmatched to an excitation wavelength is present, it will emit during atime interval associated with a pulse at the matching excitationwavelength. Accordingly, the timing or phase of signals detected from asample may identify the type of fluorophore present.

In some embodiments, a combination of fluorophore discrimination methodsmay be used. For example, in a same sample analysis, some fluorophoresmay be distinguished based on lifetime and some may be distinguishedbased on excitation wavelength—absorption band matching. Multipleexcitation wavelengths may be produced by a single laser system, asdescribed in connection with FIG. 9-6A, or by a combination of lasersystems (e.g., a gain-switched semiconductor laser and passivelymode-locked laser).

Another embodiment of a two-laser system 9-602 is depicted in FIG. 9-6B.In this system, sum-frequency generation is carried out before secondharmonic generation. For example, output pulse trains 9-120 a, 9-120 bfrom a first laser 1-110 a and second laser 1-110 b are combined at adichroic mirror DC₁ and directed to a first nonlinear optical element inwhich SFG occurs. An output pulse train may then be split (using atrichroic TC₁ or dichroic splitter) to direct at least the firstwavelength to a second nonlinear optical element where SHG occurs.Accordingly, a third pulse train 9-120 c at λ₁/2 and a fourth pulsetrain 9-120 d at λ₃ can be generated. Synchronization of the two pulsetrains may be done using a timing and electro-mechanical feedbackcontrol circuit 9-220.

V. Configurations

As may be appreciated, there may be many different configurations andembodiments of a pulsed laser 1-110 and analytical instrument 1-100 andmethods of operation. Some configurations and embodiments are givenbelow, but the invention is not limited to the listed configurations andembodiments.

(1) A mode-locked laser comprising a base plate having a maximum edgelength of not more than 350 mm, a gain medium mounted on the base plate,a first end mirror mounted on the base plate located at a first end of alaser cavity, and a saturable-absorber mirror mounted on the base plateand forming a second end mirror for the laser cavity, wherein themode-locked laser is configured to produce optical pulses by passivemode locking at a repetition rate between 50 MHz and 200 MHz.

(2) The mode-locked laser of configuration (1), further comprising abio-optoelectronic chip arranged to receive excitation pulses from themode-locked laser, wherein the bio-optoelectronic chip supportssequential incorporation of nucleotides or nucleotide analogs into agrowing strand that is complementary to a target nucleic acid,beam-steering optics arranged to direct the excitation pulses at asingle characteristic wavelength towards the bio-optoelectronic chip,and a signal processor configured to receive signals representative offluorescent emission induced by the excitation pulses at the singlecharacteristic wavelength and process the received signals to determinethe identity of four different nucleotides or nucleotide analogsincorporated into the growing strand, wherein the received signalscorrespond to the sequential incorporation of nucleotides or nucleotideanalogs into the growing strand.

(3) The mode-locked laser of (1) or (2), further comprising anadjustable mirror mount in the laser cavity that is arranged to provideonly two degrees of freedom in adjustment of a laser beam within thelaser cavity while the mode-locked laser is operating, which is the onlytwo degrees of freedom provided by an optical mount in the laser cavityfor adjusting the laser beam while the mode-locked laser is operating.

(4) The mode-locked laser of any one of (1)-(3), further comprising afirst focusing optic mounted on the base plate and located along anintracavity optical axis between the gain medium and thesaturable-absorber mirror, and a second focusing optic mounted on thebase plate and located along the intracavity optical axis between thefirst focusing optic and the saturable-absorber mirror, wherein anadjustment to the position of the first focusing optic along theintracavity optical axis changes a focal spot size of an intracavitylaser beam on the saturable-absorber mirror more than a same amount ofadjustment to the position of the second focusing optic along theintracavity optical axis.

(5) The mode-locked laser of any one of (1)-(4), further comprisingtemperature-controlling elements coupled to at least two sides of thegain medium and configured to produce an asymmetric thermal gradientacross the gain medium that steers an intracavity laser beam.

(6) The mode-locked laser of any one of (1)-(5), further comprising afirst focusing optic mounted on the base plate and located along anintracavity optical axis between the gain medium and thesaturable-absorber mirror, a second focusing optic mounted on the baseplate and located along the intracavity optical axis between the firstfocusing optic and the saturable-absorber mirror, and an intracavitybeam-steering module mounted between the first focusing optic and thesaturable-absorber mirror.

(7) The mode-locked laser of (6), further comprising a photodetectorarranged to detect an average power of the mode-locked laser, andcontrol circuitry in communication with the photodetector and theintracavity beam-steering module, wherein the control circuitry isconfigured to provide signals to realign an intracavity laser beam onthe saturable-absorber mirror based on a signal level detected by thephotodetector.

(8) The mode-locked laser of (6), further comprising a photodetector andsignal processor arranged to detect one or more characteristicsassociated with Q-switching of the pulsed laser, and control circuitryin communication with the signal processor and the intracavitybeam-steering module, wherein the control circuitry is configured toprovide signals to realign an intracavity laser beam on thesaturable-absorber mirror responsive to detecting the one or morecharacteristics associated with Q-switching.

(9) The mode-locked laser of any one of (1)-(8), further comprising aplurality of mirrors that extend a length of the laser cavity and arelocated between the gain medium and the saturable-absorber mirror, and amounting feature formed in the base plate and located between the gainmedium and the plurality of mirrors, wherein the mounting feature isconfigured to receive an end mirror or fixture to hold an end mirrorthat shortens the laser cavity.

(10) The mode-locked laser of any one of (1)-(9), further comprising atleast one trench formed in the base plate that runs in a direction ofthe intracavity optical axis and is configured to receive one or moreoptical components of the mode-locked laser.

(11) The mode-locked laser of (10), further including an integratedoptical mount formed into the base plate, the integrated optical mountcomprising two coplanar surfaces abutting opposite sides of the at leastone trench and oriented essentially perpendicular to the intracavityoptical axis, and two sloped surfaces formed on the opposite sides ofthe at least one trench and sloping towards the two coplanar surfaces.

(12) The mode-locked laser of any one of (1)-(11), further comprising aphotodetector arranged to detect optical pulses from the mode-lockedlaser, and a clock-generation circuit configured to synchronize anelectronic clock signal from a stable oscillator to optical pulsesproduced by the mode-locked laser.

(13) The mode-locked laser of any one of (1)-(12), wherein the first endmirror comprises an output coupler having a transmission betweenapproximately 10% and approximately 25%.

(14) The mode-locked laser of any one of (1)-(13), wherein a full-widthhalf-maximum duration of the optical pulses is between about 5 ps andabout 30 ps.

(15) The mode-locked laser of any one of (1)-(14), wherein a tailintensity of the optical pulses remains 20 dB below a peak intensity ofthe optical pulses after 250 ps from the peak intensity of the opticalpulses.

(16) The mode-locked laser of any one of (1)-(15), further comprising afrequency-doubling component mounted on the base plate that convertsoutput pulses from the laser from a first lasing wavelength to pulseshaving one-half the lasing wavelength.

(17) The mode-locked laser of any one of (1)-(15), further comprising afrequency-doubling component mounted on the base plate and arranged toreceive an output from the mode-locked laser, and a feedback circuitconfigured to receive a signal representative of an amount of power at afrequency-doubled wavelength delivered from the frequency-doublingcomponent to a bio-optoelectronic chip and provide a signal to changethe amount of power at a frequency-doubled wavelength based on a levelof the received signal.

(18) The mode-locked laser of (16) or (17), further comprising apolarization rotator arranged to change a polarization of the outputfrom the mode-locked laser that is delivered to the frequency-doublingcomponent, and an actuator connected to the feedback circuit thatcontrols an orientation of the polarization rotator.

(19) The mode-locked laser of any one of (1)-(18), further comprising adiode pump source module mounted to the base plate withthermally-insulating fasteners.

(20) The mode-locked laser of (19), wherein the diode pump source moduleis mounted through a hole in the base plate and is located on a side ofthe base plate opposite the laser cavity.

(21) A method for sequencing DNA, the method comprising acts ofproducing pulsed excitation energy at a single characteristicwavelength; directing the pulsed excitation energy towards abio-optoelectronic chip, wherein the bio-optoelectronic chip supportssequential incorporation of nucleotides or nucleotide analogs into agrowing strand that is complementary to a target nucleic acid; receivingsignals representative of fluorescent emission induced by the pulsedexcitation energy at the single characteristic wavelength, wherein thesignals correspond to the sequential incorporation of nucleotides ornucleotide analogs into the growing strand; and processing the receivedsignals to determine the identity of four different nucleotides ornucleotide analogs incorporated into the growing strand.

(22) The method of embodiment (21), wherein producing pulsed excitationenergy comprises producing optical pulses with a mode-locked laseroperating at a single characteristic wavelength.

(23) The method of (21), wherein producing pulsed excitation energycomprises producing optical pulses with a gain-switched laser operatingat a single characteristic wavelength.

(24) The method of any one of (21)-(23), wherein processing the receivedsignals comprises distinguishing between at least two differentfluorescent emission decay values to identify at least two differentnucleotides or nucleotide analogs of the four nucleotides or nucleotideanalogs.

(25) The method of any one of (21)-(24), further comprising producing anelectronic trigger signal that is synchronized to the pulsed excitationenergy; and providing the electronic trigger signal for timingcollection of the signals representative of fluorescent emission on thebio-optoelectronic chip.

(26) The method of (25), further comprising timing collection of thesignals representative of fluorescent emission to occur when the pulsedexcitation energy is in an off state that follows an on state.

(27) The method of any one of (21)-(26), wherein directing the pulsedexcitation energy comprises coupling the pulsed excitation energy into awaveguide on the bio-optoelectronic chip.

(28) The method of (27), wherein the coupling comprises receiving afirst feedback signal from the bio-optoelectronic chip that indicates adegree of alignment of a beam of the pulsed excitation energy to aninput port connected to the waveguide; and steering the beam based uponthe first feedback signal.

(29) The method of (27) or (28), wherein the coupling further comprisesreceiving a second feedback signal from the bio-optoelectronic chip thatindicates an amount of power delivered to the target nucleic acid; andadjusting an amount of energy in the pulsed excitation energy based uponthe second feedback signal.

(30) A bioanalytic instrument comprising a pulsed laser systemconfigured to produce optical excitation pulses at a singlecharacteristic wavelength, a receptacle for receiving abio-optoelectronic chip and making electrical connections and an opticalcoupling with the bio-optoelectronic chip, wherein thebio-optoelectronic chip supports sequential incorporation of nucleotidesor nucleotide analogs into a growing strand that is complementary to atarget nucleic acid, beam-steering optics arranged to direct theexcitation pulses towards the receptacle, and a signal processorconfigured to receive signals representative of fluorescent emissioninduced by the excitation pulses at the single characteristic wavelengthand process the received signals to determine the identity of fourdifferent nucleotides or nucleotide analogs incorporated into thegrowing strand, wherein the received signals correspond to thesequential incorporation of nucleotides or nucleotide analogs into thegrowing strand.

(31) The bioanalytic instrument of configuration (30), wherein thepulsed laser system comprises a mode-locked laser.

(32) The bioanalytic instrument of (31), wherein the mode-locked lasercomprises a base plate, a gain medium mounted on the base plate, a firstend mirror mounted on the base plate located at a first end of a lasercavity, and a saturable-absorber mirror mounted on the base plate andforming a second end mirror for the laser cavity.

(33) The bioanalytic instrument of (31) or (32), wherein the mode-lockedlaser comprises a fiber laser.

(34) The bioanalytic instrument of (31) or (32), wherein the mode-lockedlaser comprises a mode-locked laser diode.

(35) The bioanalytic instrument of (31) or (32), wherein the mode-lockedlaser comprises a diode-pumped laser having an intracavityfrequency-doubling element.

(36) The bioanalytic instrument of (30), wherein the pulsed laser systemcomprises a gain-switched laser.

(37) The bioanalytic instrument of (36), wherein the gain-switched lasercomprises a laser diode.

(38) The bioanalytic instrument of (36), wherein the gain-switched lasercomprises a laser diode, and a current driving circuit configured toprovide a bipolar current pulse to the laser diode, wherein the bipolarcurrent pulse comprises a first pulse having a first amplitude and firstpolarity that is followed by a second pulse of opposite polarity havinga second amplitude less than the first amplitude.

(39) The bioanalytic instrument of (38), wherein the driving circuitincludes a transistor coupled to a terminal of the laser diode, whereinthe driving circuit is configured to receive a unipolar pulse and applya bipolar electrical pulse to the semiconductor diode responsive toreceiving the unipolar pulse.

(40) The bioanalytic instrument of (30), wherein the pulsed laser systemcomprises a continuous-wave laser and an array of interconnected opticalswitches that modulate an output from the continuous-wave laser.

(41) The bioanalytic instrument of any one of (30)-(40), furthercomprising synchronization circuitry that controls collection of thesignals representative of fluorescent emission to occur at a time whenthe excitation pulses are in an essentially off state at thebio-optoelectronic chip.

(42) The bioanalytic instrument of (41), wherein the synchronizationcircuitry comprises a clock-generation circuit configured to synchronizea first clock signal from an electronic or electro-mechanical oscillatorto a second clock signal produced from detection of the excitationpulses and to provide the synchronized first clock signal to timedata-acquisition by the bioanalytic instrument.

(43) The bioanalytic instrument of (42), wherein the clock-generationcircuit includes automatic gain control amplification to levelamplitudes of electronic pulses generated from the optical pulses.

(44) The bioanalytic instrument of (42), wherein the clock-generationcircuit includes saturated amplification to level amplitudes ofelectronic pulses generated from the optical pulses.

(45) A bioanalytic instrument comprising a laser configured to producepulsed excitation energy at a single characteristic wavelength, and aclock-generation circuit configured to synchronize a first clock signalfrom an electronic or electro-mechanical oscillator to a second clocksignal produced from detection of optical pulses from the laser and toprovide the synchronized first clock signal to time data-acquisition bythe bioanalytic instrument.

(46) The bioanalytic instrument of configuration (45), wherein theclock-generation circuit includes automatic gain control amplificationto level amplitudes of electronic pulses generated from the opticalpulses.

(47) The bioanalytic instrument of (45), wherein the clock-generationcircuit includes saturated amplification to level amplitudes ofelectronic pulses generated from the optical pulses.

(48) The bioanalytic instrument of any one of (45)-(47), wherein theclock-generation circuit includes a phase-locked loop that locks thephase of the first clock signal to the second clock signal.

(49) The bioanalytic instrument of any one of (45)-(47), wherein theclock-generation circuit includes a delay-locked loop that locks thephase of the first clock signal to the second clock signal.

(50) A system comprising a pulsed laser, a continuous-wave laser, afirst nonlinear optical element, and a second nonlinear optical element,wherein the system is configured to produce a first pulse traingenerated from the first nonlinear optical element at a firstcharacteristic wavelength and a second pulse train from the secondnonlinear optical element at a second characteristic wavelength.

(51) The system of configuration (50), wherein the second nonlinearoptical element is in a laser cavity of the continuous-wave laser.

(52) The system of (50) or (51), wherein the second pulse train issynchronized to the first pulse train.

(53) The system of any one of (50)-(52), wherein the second pulse trainis produced by sum-frequency generation in the second nonlinear opticalelement.

(54) The system of any one of (50)-(53), wherein the first and secondcharacteristic wavelengths are between 500 nm and 700 nm.

(55) The system of any one of (50)-(54), further comprising abioanalytical instrument configured to hold a sample and beam-steeringoptics arranged to direct radiation from the first pulse train andsecond pulse train onto the sample.

(56) The system (55), wherein the bioanalytical instrument is configuredto detect emission from the sample and distinguish two or morefluorophores based on fluorescent lifetimes.

(57) A method of providing synchronized optical pulses, the methodcomprising operating a pulsed laser at a first characteristicwavelength; operating a continuous-wave laser at a second characteristicwavelength; coupling a first pulse train from the pulsed laser into alaser cavity of the continuous-wave laser; and generating a second pulsetrain at a third characteristic wavelength in the laser cavity of thecontinuous-wave laser.

(58) The method of embodiment (57), wherein generating the second pulsetrain comprises sum-frequency generation.

(59) The method of (57) or (58), further comprising frequency doubling apulse train from the pulsed laser to generate a third pulse train at afourth characteristic wavelength.

(60) The method of (59), further comprising providing the second pulsetrain and third pulse train to a bioanalytical instrument.

(61) The method of (60), further comprising exciting at least twofluorophores in a sample at the bioanalytical instrument with pulses ofthe second and third pulse trains; and distinguishing the at least twofluorophores based on fluorescent lifetimes.

(62) A system comprising a first pulsed laser, a second pulsed laser, afirst nonlinear optical element, and a second nonlinear optical element,wherein the system is configured to produce a first pulse traingenerated from the first nonlinear optical element at a firstcharacteristic wavelength and a second pulse train by sum-frequencygeneration from the second nonlinear optical element at a secondcharacteristic wavelength.

(63) The system of configuration (62), wherein the second pulse train issynchronized to the first pulse train.

(64) The system of (62) or (63), further comprising a bioanalyticalinstrument configured to hold a sample and direct radiation from thefirst pulse train and second pulse train onto the sample.

(65) The system of (64), wherein the bioanalytical instrument isconfigured to detect emission from the sample and distinguish two ormore fluorophores based on fluorescent lifetimes.

(66) The system of any of (62)-(65), further comprising a thirdnonlinear optical element, wherein the system is configured to produce athird pulse train generated from the third nonlinear optical element ata third characteristic wavelength.

(67) The system of (66), wherein the third pulse train is synchronizedto the first and second pulse trains.

(68) The system of any of (62)-(67), wherein the first, second, andthird characteristic wavelengths are between 500 nm and 700 nm.

(69) A method of providing synchronized optical pulses, the methodcomprising operating a first pulsed laser at a first characteristicwavelength; operating a second pulsed laser at a second characteristicwavelength; synchronizing the first pulsed laser to the second pulsedlaser; frequency doubling pulses from the first pulsed laser to producea first pulse train at a third characteristic wavelength; couplingpulses from the first pulsed laser and second pulsed laser into anonlinear optical element; and generating, by sum-frequency generation,a second pulse train at a fourth characteristic wavelength.

(70) The method of embodiment (69), further comprising providing thefirst pulse train and second pulse train to a bioanalytical instrument.

(71) The method of (70), further comprising exciting at least twofluorophores in a sample at the bioanalytical instrument with pulses ofthe first and second pulse trains; and distinguishing the at least twofluorophores based on fluorescent lifetimes.

(72) The method of any of (69)-(71), further comprising frequencydoubling pulses from the second pulsed laser to produce a third pulsetrain at a fifth characteristic wavelength.

(73) The method of (72), wherein the third, fourth, and fifthcharacteristic wavelengths are between 500 nm and 700 nm.

(74) A system comprising a first pulsed laser and a second pulsed laserthat includes an intracavity saturable absorber mirror, wherein thesystem is configured to direct pulses from the first pulsed laser ontothe saturable absorber mirror of the second pulsed laser.

(75) The system of configuration (74), wherein the second pulsed laseris passively mode locked.

(76) The system of (74) or (75), further comprising a first nonlinearoptical element, and a second nonlinear optical element, wherein thesystem is configured to produce a first pulse train generated from thefirst nonlinear optical element at a first characteristic wavelength anda second pulse train from the second nonlinear optical element at asecond characteristic wavelength.

(77) The system of any one of (74)-(76), further comprising abioanalytical instrument configured to hold a sample and directradiation from the first pulse train and second pulse train onto thesample.

(78) The system of (77), wherein the bioanalytical instrument isconfigured to detect emission from the sample and distinguish two ormore fluorophores based on fluorescent lifetimes.

(79) A method for mode locking two lasers, the method comprisingoperating a first pulsed laser at a first characteristic wavelength; andcoupling a pulse train from the first pulsed laser onto a saturableabsorber mirror in a laser cavity of a second pulsed laser.

(80) The method of embodiment (79), further comprising passively modelocking the second pulsed laser at a second characteristic wavelength.

(81) The method of (80) or (81), further comprising frequency doublingpulses from the first pulsed laser to produce a first pulse train at athird characteristic wavelength; and frequency doubling pulses from thesecond pulsed laser to produce a second pulse train at a fourthcharacteristic wavelength.

(82) The method of (81), further comprising exciting at least twofluorophores in a sample at a bioanalytical instrument with pulses ofthe first and second pulse trains; and distinguishing the at least twofluorophores based on fluorescent lifetimes.

(83) A pulsed laser system comprising a first mode-locked laser having afirst laser cavity configured to produce pulses having a firstcharacteristic wavelength at a first repetition rate, a second laserhaving a second laser cavity configured to produce continuous-waveradiation, a nonlinear optical element within the second laser cavity,and optical elements that direct an output from the first mode-lockedlaser into the nonlinear optical element.

(84) The pulsed laser system of configuration (83), further comprising abioanalytical instrument configured to hold a sample and direct anoutput from the second laser at a second characteristic wavelength ontothe sample.

(85) The system of (84), wherein the second characteristic wavelength isbetween 500 nm and 700 nm.

(86) The system of (84) or (85), wherein the bioanalytical instrument isconfigured to detect emission from the sample and distinguish two ormore fluorophores based on fluorescent lifetimes.

(87) The pulsed laser system of any of (83)-(86), further comprising abase structure on which the first mode-locked laser and second laser aremounted and an optical delay element located within the firstmode-locked laser that extends an optical path length of the first lasercavity to a length greater than any transverse dimension of the basestructure.

(88) The pulsed laser system of (87), wherein the optical delay elementcomprises two mirrors configured to reflect an intracavity laser beammore than two times between the two mirrors on a single pass through theoptical delay element.

(89) The pulsed laser system of (87), wherein the optical delay elementcomprises a solid block of optical material in which an intracavitylaser beam is reflected more than two times on a single pass through theoptical delay element.

(90) The pulsed laser system of (87), wherein the optical delay elementcomprises a length of optical fiber.

(91) The pulsed laser system of any of (83)-(86), further comprising abase structure on which the first mode-locked laser and second laser aremounted and a diode pump source mounted on a platform in the basestructure and arranged to excite a gain medium in the first mode-lockedlaser, wherein the diode pump source provides pump radiation betweenapproximately 450 nm and approximately 1100 nm.

(92) The pulsed laser system of (91), wherein the platform comprises anarea of the base structure that has been partially separated from thebase structure by one or more trenches extending through the basestructure.

(93) The pulsed laser system of (91), further comprising flexuralmembers connecting the platform to the base structure.

(94) The pulsed laser system of any of (83)-(90), further comprising asaturable absorber mirror configured to reflect an intracavity laserbeam of the first laser cavity, and an output coupler located at an endof the laser cavity.

(95) The pulsed laser system of any of (83)-(94), further including awavelength conversion element mounted within the base structure, whereinthe wavelength conversion element converts a lasing wavelength from thefirst mode-locked laser to a frequency-doubled output wavelength.

(96) The pulsed laser system of (95), wherein the output wavelength isbetween about 500 nm and about 700 nm and an output pulse duration isless than approximately 100 picoseconds.

(97) The pulsed laser system of (95), wherein the base structurecomprises a cavity in which the laser cavity is disposed, and an edgedimension of the base structure is no greater than about 200 mm and aheight dimension is no greater than about 60 mm.

(98) The pulsed laser system of any of (83)-(97), wherein the firstmode-locked laser is configured to lase at approximately 1064 nm and thesecond laser is configured to lase at approximately 1342 nm.

(99) The pulsed laser system of (98), wherein the nonlinear opticalelement is aligned within the second laser cavity to generate pulses ata wavelength of approximately 594 nm by sum-frequency generation.

(100) A method of producing optical pulses at multiple characteristicwavelengths, the method comprising producing optical pulses in a firstmode-locked laser having a first laser cavity at a first characteristicwavelengths operating a second laser having a second laser cavity incontinuous-wave mode at a second characteristic wavelengths injectingpulses from the first mode-locked laser into a nonlinear optical elementin the second laser cavity; and generating, by sum-frequency generation,optical pulses in the nonlinear optical element at a thirdcharacteristic wavelengths.

(101) The method of embodiment (100), wherein a same gain medium is usedin both the first mode-locked laser and the second laser.

(102) The method of (101), further comprising diode-pumping the gainmedium in each laser.

(103) The method of (101) or (102), wherein the gain medium is Nd:YVO₄.

(104) The method of any one of (100)-(103), further comprising providingoptical pulses from the second laser to a bioanalytical instrumentconfigured to hold a sample and direct the optical pulses onto thesample.

(105) The method of any one of (100)-(104), wherein the thirdcharacteristic wavelength is between 500 nm and 700 nm.

(106) The method of (104) or (105), further comprising detecting, withthe bioanalytical instrument, emission from the sample; anddistinguishing two or more fluorophores based on fluorescent lifetimes.

(107) The method of any of (104)-(106), further comprising deriving aclock signal from the optical pulses from the first mode-locked laserand providing the clock signal to the bioanalytical instrument.

(108) The method of any one of (100)-(107), wherein producing opticalpulses in a first mode-locked laser comprises passively mode-locking thefirst mode-locked laser.

(109) The method of any one of (100)-(108), wherein the firstcharacteristic wavelength is approximately 1064 nm, the secondcharacteristic wavelength is approximately 1342 nm, and the thirdcharacteristic wavelength is approximately 594 nm.

(110) The method of any one of (100)-(109), further comprising frequencydoubling optical pulses from the first mode-locked laser.

(111) A pulsed laser comprising a base structure, a diode pump sourcemounted within the base structure, and a laser cavity within the basestructure that includes a gain medium and is configured to produceoptical pulses, wherein the diode pump source and gain medium are eachmounted on a platform that is partially thermally and mechanicallyisolated from the base structure.

(112) The pulsed laser of configuration (111), further comprising anoptical delay element located within the pulsed laser cavity thatextends an optical path length of the laser cavity to a length greaterthan a transverse dimension of the base structure.

(113) The pulsed laser of (112), wherein the optical delay elementcomprises two mirrors configured to reflect an intracavity laser beammultiple times between the two mirrors.

(114) The pulsed laser of (112), wherein the optical delay elementcomprises a solid block of optical material in which an intracavitylaser beam is reflected multiple times.

(115) The pulsed laser of (112), wherein the optical delay elementcomprises a length of optical fiber.

(116) The pulsed laser of any one of (111)-(115), wherein the diode pumpsource provides pump radiation between approximately 450 nm andapproximately 1100 nm.

(117) The pulsed laser of any one of (111)-(116), further comprising apair of crossed cylindrical lenses arranged to reshape a beam from thediode pump source.

(118) The pulsed laser of any one of (111)-(117), further comprising asaturable absorber mirror within the base structure and configured toreflect an intracavity laser beam, and an output coupler located at anend of the laser cavity.

(119) The pulsed laser of any one of (111)-(118), further including awavelength conversion element mounted within the base structure, whereinthe wavelength conversion element converts a lasing wavelength from thegain medium to an output wavelength.

(120) The pulsed laser of (119), wherein the output wavelength isbetween about 500 nm and about 700 nm and an output pulse duration isless than approximately 10 picoseconds.

(121) The pulsed laser of any one of (111)-(120), further comprising abioanalytical instrument configured to hold a sample and direct anoutput from the pulsed laser at the output wavelength onto the sample.

(122) The pulsed laser of (121), wherein the bioanalytical instrument isconfigured to detect emission from the sample and distinguish two ormore fluorophores based on fluorescent lifetimes.

(123) The pulsed laser of any one of (119)-(122), wherein the basestructure comprises a cavity in which the laser cavity is disposed, andan edge dimension of the base structure is no greater than about 200 mmand a height dimension is no greater than about 60 mm.

(124) The pulsed laser of any one of (111)-(123), wherein the platformcomprises an area of the base structure that has been partiallyseparated from the base structure by one or more trenches extendingthrough the base structure.

(125) The pulsed laser of (124), further comprising flexural membersconnecting the platform to the base structure.

(126) The pulsed laser of any one of (111)-(124), wherein the basestructure comprises aluminum.

(127) The pulsed laser of any one of (111)-(126), wherein the pulsedlaser cavity includes a gain medium that supports lasing at twowavelengths and wherein the saturable absorber mirror provides saturableabsorption at the two wavelengths.

(128) The pulsed laser of (127), wherein a first lasing wavelength isapproximately 1064 nm and a second lasing wavelength is approximately1342 nm.

(129) The pulsed laser of (127) or (128), wherein the saturable absorbermirror comprises a reflector, a first multiple quantum well structurespaced a first distance from the reflector and having a first energyband-gap, and a second multiple quantum well structure spaced a seconddistance from the reflector that is greater than the first distance andhaving a second energy band-gap.

(130) The pulsed laser of (129), wherein the second energy band-gap isgreater than the first energy band-gap.

(131) The mode-locked laser of any one of (1)-(20), wherein a ratio of aminimum beam waist in the gain medium to a focused beam waist on thesaturable-absorber mirror is between 4:1 and 1:2.

(132) The mode-locked laser of any one of (1)-(20) and (131), wherein abeam radius in the gain medium is between 20 microns and 200 microns.

VI. Conclusion

Having thus described several aspects of several embodiments of a pulsedlaser, it is to be appreciated that various alterations, modifications,and improvements will readily occur to those skilled in the art. Suchalterations, modifications, and improvements are intended to be part ofthis disclosure, and are intended to be within the spirit and scope ofthe invention. While the present teachings have been described inconjunction with various embodiments and examples, it is not intendedthat the present teachings be limited to such embodiments or examples.On the contrary, the present teachings encompass various alternatives,modifications, and equivalents, as will be appreciated by those of skillin the art.

For example, embodiments may be modified to include more or feweroptical components in a laser cavity than described above. Moreover,laser cavity configurations may differ from those shown with some lasercavities have more or fewer turns or folds in the optical path.

While various inventive embodiments have been described and illustrated,those of ordinary skill in the art will readily envision a variety ofother means and/or structures for performing the function and/orobtaining the results and/or one or more of the advantages described,and each of such variations and/or modifications is deemed to be withinthe scope of the inventive embodiments described. More generally, thoseskilled in the art will readily appreciate that all parameters,dimensions, materials, and configurations described are meant to beexamples and that the actual parameters, dimensions, materials, and/orconfigurations will depend upon the specific application or applicationsfor which the inventive teachings is/are used. Those skilled in the artwill recognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific inventive embodimentsdescribed. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, inventiveembodiments may be practiced otherwise than as specifically describedand claimed. Inventive embodiments of the present disclosure may bedirected to each individual feature, system, system upgrade, and/ormethod described. In addition, any combination of two or more suchfeatures, systems, and/or methods, if such features, systems, systemupgrade, and/or methods are not mutually inconsistent, is includedwithin the inventive scope of the present disclosure.

Further, though some advantages of the present invention may beindicated, it should be appreciated that not every embodiment of theinvention will include every described advantage. Some embodiments maynot implement any features described as advantageous. Accordingly, theforegoing description and drawings are by way of example only.

All literature and similar material cited in this application,including, but not limited to, patents, patent applications, articles,books, treatises, and web pages, regardless of the format of suchliterature and similar materials, are expressly incorporated byreference in their entirety. In the event that one or more of theincorporated literature and similar materials differs from orcontradicts this application, including but not limited to definedterms, term usage, described techniques, or the like, this applicationcontrols.

The section headings used are for organizational purposes only and arenot to be construed as limiting the subject matter described in any way.

Also, the technology described may be embodied as a method, of which atleast one example has been provided. The acts performed as part of themethod may be ordered in any suitable way. Accordingly, embodiments maybe constructed in which acts are performed in an order different thanillustrated, which may include performing some acts simultaneously, eventhough shown as sequential acts in illustrative embodiments.

All definitions, as defined and used, should be understood to controlover dictionary definitions, definitions in documents incorporated byreference, and/or ordinary meanings of the defined terms.

Numerical values and ranges may be described in the specification andclaims as approximate or exact values or ranges. For example, in somecases the terms “about,” “approximately,” and “substantially” may beused in reference to a value. Such references are intended to encompassthe referenced value as well as plus and minus reasonable variations ofthe value. For example, a phrase “between about 10 and about 20” isintended to mean “between exactly 10 and exactly 20” in someembodiments, as well as “between 10±δ1 and 20±δ2” in some embodiments.The amount of variation δ1, δ2 for a value may be less than 5% of thevalue in some embodiments, less than 10% of the value in someembodiments, and yet less than 20% of the value in some embodiments. Inembodiments where a large range of values is given, e.g., a rangeincluding two or more orders of magnitude, the amount of variation δ1,δ2 for a value could be as high as 50%. For example, if an operablerange extends from 2 to 200, “approximately 80” may encompass valuesbetween 40 and 120 and the range may be as large as between 1 and 300.When exact values are intended, the term “exactly” is used, e.g.,“between exactly 2 and exactly 200.”

The term “adjacent” may refer to two elements arranged within closeproximity to one another (e.g., within a distance that is less thanabout one-fifth of a transverse or vertical dimension of a larger of thetwo elements). In some cases there may be intervening structures orlayers between adjacent elements. In some cases adjacent elements may beimmediately adjacent to one another with no intervening structures orelements.

The indefinite articles “a” and “an,” as used in the specification andin the claims, unless clearly indicated to the contrary, should beunderstood to mean “at least one.”

The phrase “and/or,” as used in the specification and in the claims,should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used shall only be interpreted as indicating exclusive alternatives(i.e. “one or the other but not both”) when preceded by terms ofexclusivity, such as “either,” “one of,” “only one of,” or “exactly oneof.” “Consisting essentially of,” when used in the claims, shall haveits ordinary meaning as used in the field of patent law.

As used in the specification and in the claims, the phrase “at leastone,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively.

The claims should not be read as limited to the described order orelements unless stated to that effect. It should be understood thatvarious changes in form and detail may be made by one of ordinary skillin the art without departing from the spirit and scope of the appendedclaims. All embodiments that come within the spirit and scope of thefollowing claims and equivalents thereto are claimed.

1-49. (canceled)
 50. A bioanalytic instrument comprising: a chassis; aprinted circuit board that mounts to the chassis; a socket on theprinted circuit board adapted to receive a bio-optoelectronic chip suchthat a plurality of electrical connections are made to thebio-optoelectronic chip; a pulsed optical source that, when operating,produces optical pulses at a repetition rate between 50 MHz and 200 MHz,wherein the pulsed optical source is assembled in a source module thatmounts to the chassis; and a clock-generation circuit configured tosynchronize a first clock signal from an electronic orelectro-mechanical oscillator to a second clock signal produced fromdetection of optical pulses from the pulsed optical source.
 51. Thebioanalytic instrument of claim 50, wherein the pulsed optical source isa passively mode-locked laser.
 52. The bioanalytic instrument of claim51, wherein a volume occupied by the source module is no greater than0.5 cubic foot.
 53. The bioanalytic instrument of claim 51, furthercomprising at least at least one optically reflecting component disposedin the source module that provide a plurality of reflections that extenda length of a laser cavity of the passively mode-locked laser.
 54. Thebioanalytic instrument of claim 51, further comprising a diode pumpsource module mounted to the source module.
 55. The bioanalyticinstrument of claim 51, wherein the diode pump source module is mountedthrough a hole in a base plate on which the passively mode-locked laseris assembled, such that heat generated by the diode pump source moduleis dissipated on a first side of the base plate that is opposite to asecond side of the base plate on which optical components of thepassively mode-locked laser are mounted.
 56. The bioanalytic instrumentof claim 51, further comprising: an intracavity beam-steering moduledisposed within a laser cavity of the passively mode-locked laser; aphotodetector and signal processor configured to detect one or morecharacteristics associated with Q-switching of the passively mode-lockedlaser; and control circuitry in communication with the signal processorand the intracavity beam-steering module, wherein the control circuitryis configured to provide signals to realign an intracavity laser beam inresponse to detecting the one or more characteristics associated withQ-switching.
 57. The bioanalytic instrument of claim 51, wherein afull-width half-maximum duration of the optical pulses is between about5 ps and about 30 ps.
 58. The bioanalytic instrument of claim 50,further comprising signal processing circuitry configured to: receivesignals from the bio-optoelectronic chip that were generated in responseto optical excitation of fluorophores at the bio-optoelectronic chip bya single characteristic wavelength; and determine one type of signalthat the received signals are indicative of from among a plurality ofdifferent signal types that are generated in response to opticalexcitation of fluorophores at the bio-optoelectronic chip by the singlecharacteristic wavelength.
 59. The bioanalytic instrument of claim 50,wherein the clock-generation circuit is mounted to the source module.60. The bioanalytic instrument of claim 50, wherein an output from theclock-generation circuit provides the synchronized first clock signal tothe bioanalytic instrument to time data-acquisition at thebio-optoelectronic chip.
 61. The bioanalytic instrument of claim 60,wherein signals are collected at the bio-optoelectronic chip at a timewhen excitation pulses from the pulsed optical source are in anessentially off state at the bio-optoelectronic chip.
 62. Thebioanalytic instrument of claim 50, wherein the clock-generation circuitincludes automatic gain control amplification to level amplitudes ofelectronic pulses generated from the optical pulses.
 63. The bioanalyticinstrument of claim 50, wherein the clock-generation circuit includessaturated amplification to level amplitudes of electronic pulsesgenerated from the optical pulses.
 64. The bioanalytic instrument ofclaim 50, wherein the clock-generation circuit includes a phase-lockedloop that locks the phase of the first clock signal to the second clocksignal.
 65. The bioanalytic instrument of claim 50, wherein theclock-generation circuit includes a delay-locked loop that locks thephase of the first clock signal to the second clock signal.
 66. Thebioanalytic instrument of claim 50, further comprising a beam-steeringmodule that mounts to the chassis, wherein the beam-steering moduleincludes a first optical component arranged to adjust an incident angleof a beam from the pulsed optical source on the bio-optoelectronic chipessentially without adjusting a position of the beam on thebio-optoelectronic chip.
 67. The bioanalytic instrument of claim 66,further comprising circuitry configured to: receive a signal from thebio-optoelectronic chip indicative of power coupled into a waveguide ofthe bio-optoelectronic chip; and control the orientation of at least oneoptical component in the beam-steering module to change an amount ofpower coupled into the waveguide.
 68. The bioanalytic instrument ofclaim 66, wherein the printed circuit board attaches to thebeam-steering module so as to reduce relative motion between thebeam-steering module and the socket that receives the bio-optoelectronicchip.
 69. The bioanalytic instrument of claim 66, wherein thebeam-steering module comprises three stepper motors with rotatableshafts adapted to rotate optical elements in the beam-steering module,wherein axes of the rotatable shafts all lie in essentially the sameplane.